Transcription II_Adv Mol Biol_MSc_2023-24.pdf

GENE REGULATION
Year I MSc in Biotechnology
Year I MSc in Molecular Biology
Year I MSc in Bioinformatics
Tesfaye Sisay (DVM, MSc, PhD)
Prof. in Health Biotechnology

i. Regulation at Transcription
Transcription often is controlled at the stage of initiation
Transcription is not usually controlled at elongation
It may be controlled at termination to determine whether RNA polymerase is
allowed to proceed past a terminator to the gene(s) beyond
In eukaryotic cells
- processing of the RNA product may be regulated at the stages of
modification, splicing, transport, or stability
In bacteria
- an mRNA is in principle available for translation as soon as (or even while)
it is being synthesized, and these stages of control are not available

The Operon
The Jacob–Monod operon model of gene regulation
1959 : François Jacob and Jacques Monod
The operon model introduced the novel concept of regulatory genes that
code for products that control other genes
Jacob and Monod provided the basic concept for how transcription is
controlled in bacteria
Jacques Monod. A key
scientist in discovering
principles of gene regulation

The Jacob–Monod operon model. The Jacob–Monod operon model for the control of the synthesis of
sugar-metabolizing enzymes predicted the existence of a repressor molecule that is produced from a
regulator gene (R) and binds to an operator site (O) (called an operator “gene” in the original model),
thereby stopping the expression of the structural genes (A, B) that follow the operator site. The repressor
also binds an inducer (metabolite) that lowers the affinity of the repressor for the operator and allows expression of
the structural genes. The model also predicted the existence of an RNA intermediate (messenger) in protein
synthesis.

Jacob and Monod’s model
- based on experimental observations in bacteria and phages
Monod: investigated how the enzyme β-galactosidase was produced in
bacterial cells only when bacteria needed this enzyme to use the sugar
lactose
Jacob: studied how phage lambda (λ) could be induced to switch from the
lysogenic (quiescent) state to the lytic state
Their collaborative work:
regulation of the three enzymes involved in lactose metabolism
- occurs at the level of gene expression and
- the inducer (lactose) acts on a repressor of transcription

The essential features of gene regulation
- are similar in both lac operon induction and λ switch
The lac operon - an example of negative control of the enzymes involved
in lactose metabolism
Initially Jacob and Monod proposed that all gene regulation occurred by
negative control
Now the lac operon is also known to be regulated by positive control
under certain environmental conditions

Characterization of the Lac repressor
The Jacob–Monod model for gene regulation also proposed the existence
of a repressor protein
1966 – 1972: both Lac and λ repressors were shown to be proteins
- They bind to operator DNA adjacent to the promoter and inhibit the
capacity of RNA polymerase to transcribe
1966: Walter Gilbert and Benno Müller-Hill
- detected and isolated lac operon repressor
In vitro-binding assay - determine whether Lac repressor bound to
operator DNA

Before the advent of gene cloning techniques
- studies of bacterial genes had to rely on bacteriophage variants that had
incorporated pieces of bacterial DNA
- a phage strain which included lac operon DNA was available
Gilbert and Müller-Hill mixed:
- radioactively labeled purified Lac repressor + phage phi (φ) 80 DNA that
had incorporated the lac operator, or
- radioactively labeled purified Lac repressor + phage φ80 lacking the lac
operator

Centrifugation of these mixtures
- to separate the large DNA molecules that sedimented rapidly from the
small protein molecules that sedimented more slowly
Radioactive Lac repressor sedimented with lac DNA
- but not with the control DNA lacking the lac operator
 the Lac repressor protein binds operator DNA

Gilbert and Müller-Hill’s
experiment demonstrating that
the Lac repressor binds operator
DNA
After glycerol gradient
centrifugation, radioactively labeled
Lac repressor sedimented with
phage φ80 DNA, which had the lac
operator, but not with φ80 DNA,
which lacked the lac operator.
Radioactive
lac repressor
Phage φ 80 DNA
with lac operator
Phage φ 80 DNA
without lac operator

The lac repressor binds to operator DNA. A radioactive tag is attached to the lac repressor protein so it can be followed in the
experiment. (a) When repressor protein from lacl cells was purified and mixed with DNA containing the lac operator (on bacterial
virus DNA), the protein cosedimented with the DNA. (b) When wild-type repressor was mixed with DNA containing a mutant operator
site, no radioactivity sedimented with the DNA.

At about the same time, Mark Ptashne et al.
- isolated the λ cI repressor for λ phage operons
Then, more sophisticated techniques have confirmed the sequence-
specific binding of the Lac repressor to the lac operator
- DNase I footprinting
- electrophoretic gel mobility shift assays (EMSA)
There are now crystal structures of the Lac repressor and many other
DNA-binding proteins

Domains of repressor protein. X-ray crystallographic data
enable the construction of a model of repressor structure that
shows a region to which operator DNA binds and another
region to which inducer binds.
DNA recognition sequences by helix-turn helix motif. A protein motif
that has the shape of a helix-turn-helix ( helixes shown here
inside a cylindrical shape ) fits into the major groove of the DNA
helix. Specific amino acids within the helical region of the
protein recognize a particular base sequence in the DNA.

DNase footprint shows where
proteins bind. DNase footprint
establishes the region to which a protein
binds. A partial digestion with DNase I
produces a series of fragments. If a protein
is bound to DNA, DNase cannot digest at
sites covered by the protein. Gel
electrophoresis of digested products shows
which products were not generated and
indicates where the protein binds.

Lactose (lac) operon regulation
In bacteria, genes are organized into operons
An operon - a unit of bacterial gene expression and regulation
- including structural genes and control elements in DNA recognized by
regulatory gene products
The genes in an operon are transcribed from a single promoter
- to produce a single primary transcript (pre-mRNA) or “polycistronic
mRNA”

The nematode worm C. elegans differs from all other eukaryotes
- has ~15% of its genes grouped in operons
However, each C. elegans pre-mRNA is processed into a separate mRNA
for each gene rather than being translated as a unit
Some of the genes in C. elegans operons appear to be involved in the
same biochemical function, but this may not be the case for most

Bacteria need to respond swiftly to changes in their environment
- to switch from metabolizing one substrate to another quickly and
energetically efficiently
When glucose is abundant, bacteria use it exclusively as their food
source, even when other sugars are present
However, when glucose supplies are depleted, bacteria have the ability to
rapidly take up and metabolize alternative sugars, such as lactose
The process of induction – the synthesis of enzymes in response to the
appearance of a specific substrate
– a widespread mechanism in bacteria and single-celled eukaryotes, such
as yeast

The lactose (lac) operon in E. coli is regarded as a paradigm for
understanding bacterial gene expression
Features of the lac operon illustrate basic principles of gene regulation
that are universal
 There is a constitutively active RNA polymerase that alone works with
a certain frequency
 The transcriptional activator - increases the frequency of initiation by
recruiting the RNA polymerase to the gene promoter
 The transcriptional repressor - decreases the frequency of initiation
by excluding the polymerase
Both the repressor and activator
- DNA-binding proteins that undergo allosteric modifications

Lac operon induction
The lac operon consists of three structural genes:
lacZ, lacY, lacA
 LacA - encodes for a transacetylase - removes toxic thiogalactosides
that get taken up by the permease from the cell
 LacZ gene - encodes β-galactosidase
- cleaves lactose into galactose and glucose, both of which are used by
the cell as energy sources
 LacY - encodes lactose permease
- membrane-bound protein that is part of the transport system to bring β-
galactosides such as lactose into the cell

Lactose utilization in an
E. coli cell. Lactose passes
through the membranes of
the cell via an opening
formed by the lactose
permease protein. Inside
the cell, b-galactosidase
splits lactose into galactose
and glucose.

LacY may not be absolutely required for lactose metabolism
- mutations in lacZ create cells which cannot use lactose
- lacA mutants still can metabolize lactose
Upstream of the lac operon is the regulatory gene that codes for the Lac
repressor
The Lac repressor is constitutively transcribed under control of its own
promoter

In the absence of lactose
- the Lac repressor binds as a tetramer to the operator DNA sequence
The lac operator sequence overlaps with the promoter region
 the Lac repressor blocks RNA polymerase from binding to the
promoter
 transcription of the lac operon structural genes is repressed

Regulatory protein binding sites overlap. The lac repressor bound to the operator prevents RNA polymerase from
binding. The binding sites for RNA polymerase and repressor (determined by DNase digestion experiments) show
that there is overlap between the two sites.

Lac operon regulation by glucose and lactose

Lac operon regulation by glucose and lactose. (A) The components of the lac operon. The Lac
repressor protein is encoded by the repressor gene (I), which is under control of its own promoter (PI). The
Lac repressor binds to the lac operator (O) as a tetramer. The start of transcription (+1) is indicated. The
catabolic activator protein (CAP) site is the DNA-binding site for the activator protein CAP. The CAP
protein is encoded by a separate gene distant from the lac operon. It binds DNA as a dimer. The lac
operon structural genes are under control of the lac promoter (PLac). (B and C) Transcription of the lac
operon is repressed in the absence of lactose, whether glucose is absent (B) or present (C). Under both
conditions, the Lac repressor protein binds the operator and excludes RNA polymerase. (D) In the
presence of both glucose and lactose, RNA polymerase binds the lac promoter very poorly, resulting in a
low (basal) level of transcription.

lac operon induction
- presence of lactose and absence of glucose
The real inducer is an alternative form of lactose called allolactose
Because repression of the lac operon is not complete, there is always a very
low level of the lac operon products present (< 5 molecules per cell of β-
galactosidase)
- so some lactose can be taken up into the bacterium and metabolized
When β-galactosidase cleaves lactose to galactose plus glucose it
rearranges a small fraction of the lactose to allolactose

Structures of lactose, allolactose, and the lactose analog IPTG

Structures of lactose, allolactose, and the lactose analog IPTG. The enzyme β-galactosidase
hydrolytically cleaves lactose into glucose and galactose. A side reaction carried out by the enzyme
rearranges lactose to form the inducer, allolactose. Note the change in the galactosidic bond from β-1,4 in
lactose, to β-1,6 in allolactose. β-galactosidase cannot metabolize isopropylthiogalactoside (IPTG), a
sulfur-containing analog of lactose that is used in molecular biology research.

Even a small amount of the inducer is enough to start activating the lac
operon
Upon binding allolactose
- the Lac repressor undergoes a conformational (allosteric) change
- alters its operator-binding domain
This allosteric change reduces its DNA-binding affinity, thereby relieving
lac repression
Not only is there release from repression, there is also activation of
transcription

At a site distant from the lac operon is the gene that encodes catabolic
activator protein (CAP) (or, CRP- cyclic AMP receptor protein)
CAP - binds to the DNA sequence within the lac operon called the CAP site
Recruitment of RNA polymerase requires the formation of a complex of
CAP, polymerase, and DNA
- cooperative binding of proteins to DNA
CRP–cAMP dimer. CRP–cAMP
binds as a dimer to a regulatory
region.

Positive regulation by CRP–
cAMP. High level expression of
the lac operon requires that a
positive regulator, the CRP–cAMP
complex, be bound to the
promoter region.

CRP-cAMP interaction. The CRP–cAMP complex contacts RNA polymerase directly to help in
transcription initiation.

When the lac operon is activated
- RNA polymerase begins transcription from the promoter and transcribes a
common mRNA for the three structural genes
The mRNA has a start (AUG) codon and stop codon for each protein
The ribosome binds to the 5′ end of the mRNA and begins translation
When it reaches the stop codon at the end of the β-galactosidase-coding
region the ribosome may detach
But most continue on to the next coding region, to synthesize the permease,
followed by the transacetylase

Lac operon regulation by
glucose and lactose
Within 8 minutes after induction, approximately 5000 molecules of β-galactosidase per
cell are produced

(E) When lactose is present and glucose is absent the lac operon is induced. Binding of
the inducer allolactose changes the conformation of the Lac repressor and alters
its operator-binding domain. CAP, along with its small molecule effector cAMP, recruits
RNA polymerase and binds the CAP site and transcription is stimulated 20–40-fold. The
structural genes are transcribed as a polycistronic mRNA that is then translated using
the start and stop codon for each individual protein.

Basal transcription of the lac operon
The lac operon is subject to both positive and negative regulation
The lac operon is transcribed if and only if lactose is present in the medium
This signal is almost entirely overridden by the simultaneous presence of
glucose - a more efficient energy source than lactose
When provided with a mixture of sugars, including glucose, the bacteria
use glucose first

Glucose exerts its effect by decreasing synthesis of cAMP, which is
required for the activator CAP to bind DNA
Without the cooperative binding of CAP, RNA polymerase transcribes the
lac genes at a low level
- the basal level
The basal level is 20–40-fold lower than activated levels of transcription
So long as glucose is present, operons such as lactose are not transcribed
efficiently
Only after exhausting the supply of glucose does the bacterium fully turn on
expression of the lac operon

Mode of action of transcriptional regulators
Based on studies on many bacterial operons to understand the mode of
action of transcriptional regulatory proteins
The proteins are modular - consist of domains with distinct functions:
- for DNA binding and protein–protein interactions
 In many cases, these regulatory proteins bind to DNA in a cooperative
fashion with other proteins
 Allosteric modification plays a key role in regulation of their activities
 Distant DNA regulatory sites are brought in close proximity
- through cooperative protein–protein interactions that cause DNA
looping

Domains of repressor protein. X-ray crystallographic
data enable the construction of a model of repressor
structure that shows a region to which operator DNA
binds and another region to which inducer binds.
DNA recognition sequences by helix-turn helix motif. A
protein motif that has the shape of a helix-turn-helix (
helixes shown here inside a cylindrical shape ) fi ts into
the major groove of the DNA helix. Specific amino acids
within the helical region of the protein recognize a
particular base sequence in the DNA.

i. Cooperative binding of proteins to DNA
- plays a central role in gene regulation in both prokaryotes and eukaryotes
The effects are mediated by protein–protein and protein–DNA interactions
Ex. CAP has two major functional domains:
- a DNA binding domain and
- an “activating region” - contacts the RNA polymerase
- The distinct functions of these domains have been characterized by
mutagenesis studies

The CAP-activating region - interacts directly with the C-terminal domain
of one of the α-subunits of RNA polymerase
Through this interaction, CAP recruits RNA polymerase to the promoter
When CAP and RNA polymerase are both present their binding sites are
much more likely to be occupied
- they help each other bind to DNA
CRP-cAMP interaction. The
CRP–cAMP complex contacts
RNA polymerase directly to
help in transcription initiation.

ii. Allosteric modifications and DNA binding
Both CAP and the Lac repressor bind to their DNA sites using a similar
structural motif - a helix-turn-helix (HTH)
Each HTH has one α-helix - the recognition helix
- inserts into the major groove of DNA
The side chains of amino acids exposed along the recognition helix make
sequence-specific contacts with functional groups exposed on the base
pairs
A second α-helix lies across the DNA
- It helps position the recognition helix and strengthens the binding affinity

Domains of repressor protein. X-ray
crystallographic data enable the construction
of a model of repressor structure that shows a
region to which operator DNA binds and
another region to which inducer binds.
DNA recognition sequences by helix-turn helix
motif. A protein motif that has the shape of a
helix-turn-helix ( helixes shown here inside a
cylindrical shape ) fi ts into the major groove of
the DNA helix. Specific amino acids within the
helical region of the protein recognize a
particular base sequence in the DNA.

Specific interactions provide the molecular basis for binding specificity
and target recognition – between:
- the hydrogen bond donors and acceptors of the protein-binding site
and
- those of the base pairs in the major and minor grooves of the DNA double
helix
Electrostatic interactions support & stabilize the binding:
- negatively charged phosphates of the sugar–phosphate backbone of
the DNA and
- the basic amino acid residues that surround the binding site of the Lac
repressor

Differences in the residues along the outside of the recognition helix
largely account for differences in the DNA-binding specificities of regulators
The HTH motif is the predominant DNA recognition motif found among
E. coli transcriptional regulatory proteins
A somewhat modified form is found in eukaryotes in homeodomain proteins

The allosteric change undergone by CAP upon binding cAMP increases its
ability to bind DNA
In contrast, the allosteric change in the Lac repressor upon binding the
inducer allolactose (or the lactose analog, IPTG) decreases its ability to bind
DNA
The addition of IPTG - causes a conformational change in the N-terminal
domain of the Lac repressor dimer
- leads to separation of the hinge helices
The HTH DNA-binding motifs become disordered and dissociate from the
major groove binding site

The CAP–DNA complex. (A) Model showing the helix-turn-helix (HTH) DNA-binding
motif of one subunit of CAP. The recognition helix (F) contacts the DNA in the major
groove. The three α-helices are depicted as cylinders and β-pleated sheets as flat
ribbons. (B) Ribbon model of the CAP–DNA complex model derived from a co-crystal
structure. The inset shows the location of the two bound cAMP molecules (red). The
DNA (green) is bent by about 90° overall. The protein dimer (blue and gray subunits) is
held together through interaction between two long α-helices.

Lac repressor–DNA recognition

Lac repressor–DNA recognition. (A) Allosteric changes in the Lac repressor. A ribbon
diagram of the Lac dimer–DNA complex is shown in the darker brown shade; the Lac–
IPTG complex is shown in the lighter brown shade. The addition of IPTG (a lactose
analog) causes the hinge helices in the repressor to move apart. The helix-turn-helix
(HTH) DNA-binding motifs become disordered and move out of the major groove binding
site. The cartoons below the structures summarize these changes. The left side shows a
dimer of Lac repressor bound to IPTG (asterisk). A number of salt bridges (gray symbols)
exist between the dimers but the HTH domains are far apart and the hinge helices are
not formed. The right side shows a dimer of the Lac repressor–DNA complex. The salt
bridges are broken, the hinge helices form, and the HTH domain becomes ordered and
binds DNA.

(C) Cartoon of the Lac tetramer
bound to an upstream auxiliary
operator and the primary operator
DNA sequence (space-filling
representation), forming a DNA
loop in between (not drawn to
scale). The Lac repressor is a
tethered dimer of dimers.

iii. DNA looping
- widely used in gene regulation
- Allows multiple proteins to interact with RNA polymerase
- some from adjacent sites and some from distant sites
The cooperative binding of proteins to multiple DNA-binding sites
- increases their effective binding constants and
- allows regulatory proteins to function at very low concentrations within the
cell

Ex. the arabinose operon - controls use of arabinose
- the regulatory protein AraC acts both as a repressor and activator of
transcription
Arabinose binds to AraC
- changes the shape of the activator so that it binds as a dimer to two
regulatory sequence half sites
This places one monomer of AraC close to the promoter from which it can
activate transcription
The promoter is also further activated by CAP recruitment of RNA
polymerase

In the absence of arabinose
- the AraC dimer folds into a different conformation and one monomer
binds to a half site 194 bp upstream
When AraC binds in this manner, the DNA between the two sites forms a
loop
The DNA loop sterically blocks access of RNA polymerase to the
promoter

AraC acts as both a repressor and
an activator. The AraC protein can
bind to sites araI 1 , araI 2 , and
araO. (a) When no arabinose is
present, the binding of AraC to araO
and the araI 1 sites causes looping
of the DNA and prevents RNA
polymerase from transcribing the
genes. (b) When arabinose
(inducer) is present, AraC binds to
araI 1 and araI 2 but not to araO.
RNA polymerase interacts with AraC
at the araI sites and transcribes the
genes.

Regulation of the arabinose
operon. (A) The domain structure is
shown for one subunit of the dimeric
regulatory protein AraC. AraC acts
as a repressor in the absence of
arabinose (B) and as an activator in
the presence of arabinose

(C). AraC binds to 17 bp half sites of similar sequence called O2, I1, and I2. Another regulatory element O1
is formed of two half sites (O1L and O1R) that bind two subunits of AraC. AraC and the arabinose operon
structural genes (araB, araA, and araD) are transcribed in opposite directions from the pC and pBAD
promoters, respectively (arrows show the direction of RNA polymerase movement). At pC and O1, the RNA
polymerase and AraC compete for binding. There is a single CAP-binding site required for the activation of
the pBAD promoter, but not the pC promoter. The araBAD structural genes are only expressed in the
presence of arabinose. The araBAD operon encodes the enzymes responsible for converting arabinose
into xylulose-5-phosphate. In the absence of arabinose, two AraC proteins bind to both O2 and I1 and then
to each other. This results in the formation of a loop in the DNA. The presence of this loop blocks activation
of the pBAD promoter by RNA polymerase and thus no ara operon expression occurs.

DNA looping was first predicted upon the discovery that the negative control
element araO2 was nearly 200 bp upstream of all the sequences required
for positive control
Subsequently, looping was found to occur in a number of other prokaryotic
systems, including the lac operon
In addition, DNA looping is now known to explain how eukaryotic
enhancers act from a distance
The Lac repressor binds DNA as a tetramer, but functions as “a dimer of
dimers”

lac repressor tetramer binds to two sites. The lac repressor is a tetramer. For
simplicity we previously showed a single repressor object binding to one operator site,
but in reality, there are two identical LacI subunits that bind to each operator site. Two of
the subunits bind to the sequence in one operator site (O 1 ), and the other two subunits
bind to a second operator (either O 2 or O 3 ).
Each operator is contacted by only two
of the four subunits
The other two subunits within the
tetramer can bind to one of two other
lac operators, located 400 bp
downstream and 90 bp upstream of the
primary operator adjacent to the
promoter

In each case, the intervening DNA loops out to accommodate the reaction
Schematic representation of the helical-twist experiment that demonstrated DNA looping. When
half-integral turns were introduced in the DNA between the O2 and I1 half sites in the arabinose operon,
this interfered with repression of pBAD in the absence of arabinose. There was a 5–10-fold elevation of the
basal level of transcription from the operon. Introduction of integral numbers of turns did not interfere with
this repression.

The HU (heat-unstable nucleoid protein) and IHF (integration host
factor) proteins
- are often required as positive factors in gene expression
Both are heterodimers- consist of two different subunits
The four subunits (two from each protein) are all similar in sequence and 3-
D structure
- HU and IHF can, to some extent, substitute for each other
Action at a Distance and DNA Looping

They help in integration, inversion and recombination events by bending
DNA into the appropriate conformation
They also affect the expression of certain genes that need the DNA in
their upstream regions to be bent, in order to be transcribed
A variety of other accessory proteins, activator proteins, and repressor
proteins are also involved in the looping of DNA
HU is relatively nonspecific
IHF is more specific
Both are examples of proteins that are involved in bending DNA

Nitrogen metabolism
Many genes involved for this process require the alternative sigma factor
RpoN (= NtrA = s54) for their transcription
In addition, they are regulated by activator proteins that bind far
upstream
Most activator proteins bind just upstream of the promoter and make
direct contact with RNA polymerase, so helping it bind to the promoter
For the activators of RpoN-dependent promoters to touch the RNA
polymerase, the DNA must form a loop
The bend results from IHF binding between the promoter and activator
sites

Looping of DNA in RpoN-dependent Promoter

Looping of DNA in RpoN-dependent Promoter. The sites for binding of transcription factors and the
alternative sigma factor, RpoN, are shown in A. The IHF protein induces a bend in the DNA, which brings
the NtrC sites close to the binding site for the alternative sigma factor RpoN. Because the DNA loops
around, the RNA polymerase can be bound by two sets of NtrC dimers as well as by the RpoN protein.

Control of gene expression by RNA
RNA frequently plays a more direct role in controlling gene expression
Differential folding of RNA plays a major role in transcriptional
attenuation in bacteria
Other RNA-based regulatory mechanisms have been discovered more
recently:
- pathways involving riboswitches

i. Differential folding of RNA: transcriptional attenuation of the
tryptophan operon
Regulation of the tryptophan (trp) operon in bacteria
During transcription of the leader region of the trp operon
- a domain of the newly synthesized RNA transcript can fold to form either
of two competing hairpin structures:
an antiterminator or
a terminator
The leader RNA preceding the antiterminator contains a 14 nt coding
region, trpL - includes two tryptophan codons

When bacterial cells have adequate levels of tryptophan
- the leader peptide (trpL) is synthesized
- the terminator forms in the leader transcript, and transcription is
terminated
When cells are deficient in tryptophan
- the ribosome translating trpL stalls at one of these tryptophan codons
This stalling allows the downstream sequence to fold, forming an
antiterminator structure that prevents formation of the competing terminator
Termination is blocked, allowing transcription of sequences encoding the
structural genes involved in tryptophan biosynthesis

In addition, transcription initiation in the trp operon is also controlled by
more conventional means
A tryptophan-activated repressor binds to operator sites located within
the trp promoter region
- blocks access of RNA polymerase to the trp promoter
The major effect of transcription is through trp repression
The effect of attenuation on transcription is about 10-fold
These two regulatory mechanisms in combination
- allow about a 600-fold range of transcription levels of the trp operon
structural genes

Transcriptional attenuation of the E. coli trp operon. (A) Repression mediated by differential folding
of RNA. Termination: when the amino acid tryptophan is abundant in the cell, the ribosome translating
trpL does not stall at the tandem Trp codons in trpL and rapidly reaches the trpL stop codon. The terminator
hairpin forms in the transcript, resulting in the termination of transcription. The structural genes for the enzymes
involved in tryptophan biosynthesis (trpEDCBA) are not transcribed. Antitermination: a deficiency in charged
tRNATrp stalls the translating ribosome at one of the two tandem Trp codons in trpL. This stalling allows the
antiterminator hairpin to form, which prevents terminator formation. Transcription of the structural gene coding regions
takes place.

(B) Protein-mediated repression. In the absence of tryptophan, the genes encoding the biosynthetic
enzymes for tryptophan are transcribed and translated. When enough tryptophan has been produced, the
tryptophan binds to the dimeric Trp repressor protein, inducing a conformational change that enables it to
bind the trp operator, thereby blocking access of RNA polymerase to the trp promoter.

ii. Riboswitches
- specialized domains within certain mRNAs that act as switchable
“on–off ” elements
- selectively bind metabolites and control gene expression without the
need for protein transcription factors
RNA can function as a sensor for signals
- temperature, salt concentration, metal ions, amino acids, and other
small organic metabolites
Riboswitches are widespread in bacteria

However, only one type of riboswitch has been found in eukaryotes so far:
- a thiamine pyrophosphate-sensing riboswitch
- present in plants and fungi
Riboswitches are typically found in the 5′ untranslated regions of mRNAs
Most riboswitches can be divided roughly into two structural domains:
- an aptamer (RNA receptor) and
- an “expression platform”

The term expression platform is just another name for the type of
attenuation mechanism described for the trp operon
The expression platform domain has the potential to form alternative
antiterminator and terminator hairpins
The aptamer domain - selectively binds to the target metabolite
The expression platform - converts metabolite-binding events into
changes in gene expression via changes in RNA folding that are
brought about by ligand binding

Mechanisms of riboswitch gene control. (A) Most riboswitches are comprised of a < 100 nt aptamer
(RNA receptor) domain and an expression platform which has inducible secondary structures.
Transcription control involves metabolite binding and stabilization of a specific conformation of the aptamer
domain that precludes formation of a competing antiterminator stem in the expression platform. This allows
formation of a terminator stem, which prevents the full-length mRNA from being synthesized. In contrast,
control of translation is accomplished by metabolite- or temperature-induced structural changes that
sequester the ribosome-binding site (RBS), thereby preventing the ribosome from binding to the mRNA.
Riboswitches as versatile gene control elements.

Mechanisms of riboswitch gene control.

(B) A ribozyme riboswitch. The GlmS enzyme, which is involved in the synthesis of
GlcN6P (green hexagon), is encoded by the glmS mRNA, which also contains a
ribozyme sequence. In the presence of GlcN6P, the ribozyme cleaves its own mRNA.
Self-destruction of the glmS mRNA inhibits further production of GlcN6P. (Inset) (i)
Chemical structures and names of various compounds used to explore the substrate
specificity of the glmS ribozyme. (ii) Ribozyme cleavage assays using the glmS RNA and
various compounds showed that significant cleavage (Clv) only occurred in the presence
of GlcN6P and Mg2+. 5′-[32P]-labeled precursor (Pre) RNAs were incubated for the
times indicated in the absence (−) or presence of 200 μM effector as noted for each lane.
Self-cleaved RNA was separated from precursor RNA by polyacrylamide gel
electrophoresis and visualized by autoradiography.

Metabolite sensors
The genes controlled by riboswitches often encode proteins involved in the
biosynthesis or transport of the metabolite being sensed
In most cases, the metabolite-sensing riboswitch is used as a form of
feedback inhibition
Binding of the metabolite to the riboswitch serves as a genetic “off ”
switch and decreases the expression of the gene products used to make
the metabolite
Repression occurs either by:
- terminating transcription to prevent the production of full-length mRNAs, or
- preventing translation initiation once a full-length mRNA has been made

For transcriptional control in the absence of a bound metabolite, an
antiterminator RNA secondary structure is formed
When a metabolite binds, a competing stem structure is formed that acts as
a transcription terminator
For control of translation initiation, the bound aptamer blocks the ribosome-
binding site
In some rare cases, the riboswitch acts as a genetic “on” switch to activate
gene expression
Ex. binding of adenine or glycine to the adenine-sensing or glycine-sensing
riboswitches, respectively, promotes mRNA transcription by preventing
formation of a terminator stem

Anti-Termination as a Control Mechanism
Anti-termination factors - proteins that prevent termination at specific
sites
The RNA polymerase continues on its way and transcribes the region of
DNA beyond the terminator
This mechanism is common in bacteriophages but is also found for a few
bacterial genes
Anti-termination factors attach themselves to the RNA polymerase
before it reaches the terminator
The recognition sequences for anti-termination are found in the DNA well
upstream of the terminator

As the RNA polymerase passes by, the anti-termination factors are loaded
on
They remain attached and allow the RNA polymerase to travel past the
stem and loop region of the terminator without pausing
Consequently, termination is suppressed

Operation of Anti-Termination Factor

Operation of Anti-Termination Factor
A) Transcription of the DNA begins with the RNA polymerase bound to the promoter. B)
In the absence of an anti-terminator factor, the RNA polymerase reaches the terminator
region and drops off, having transcribed a short RNA. C) In the presence of an anti-
termination factor, the RNA polymerase binds the factor when it reaches the recognition
site. The factor allows the RNA polymerase to transcribe through the termination site.

Anti-termination in E. coli involves Nus proteins
NusA protein is probably attached to the core RNA polymerase shortly
after the sigma factor is lost just after
initiation
NusA itself actually promotes termination
- by increasing the duration of pauses at hairpin structures
NusA and sigma cannot both bind to the core enzyme simultaneously
As long as RNA polymerase is attached to DNA, the Nus proteins cannot be
dislodged
However, addition of sigma displaces NusA from free RNA polymerase

Thus, RNA polymerase cycles between initiation mode (with sigma
bound) and termination mode (with NusA bound)
Sigma NusA Cycle of RNA Polymerase

RNA polymerase needs the sigma subunit to recognize and bind to the promoter. Once
RNA polymerase moves forward and starts transcribing, it releases sigma and picks up
NusA protein. After termination, NusA protein is displaced by sigma.

The other Nus proteins are involved in anti-termination
NusB plus RpsJ (=S10 = NusE) - attached to the RNA polymerase as it
passes a “boxA” anti-termination sequence
NusG protein probably helps in loading
The presence of NusA is required for NusB plus RpsJ to
bind to RNA polymerase
The best known genes in E. coli that show anti-termination are the rrn
genes for synthesis of rRNA

Operation of Anti-Termination in E. coli rrn Genes
As RNA polymerase passes the boxA sequences, NusG protein loads NusB plus NusE
(= S10 = RpsJ) onto the polymerase. These Nus proteins prevent premature termination.

Transcription regulation in eukaryotes
Long-range regulatory elements
Protein-coding genes of multicellular eukaryotes typically contain additional
regulatory DNA sequences
- can work over distances of 100 kb from the gene promoter
- are instrumental in mediating the complex patterns of gene expression
in different cell types during development
Such long-range regulation is not generally observed in yeast

Long-range regulatory elements in multicellular eukaryotes include:
- enhancers and silencers
- Insulators
- locus control regions (LCRs), and
- matrix attachment regions (MARs)

i. Enhancers and silencers
A typical protein-coding gene is likely to contain several enhancers which
act at a distance
- usually 700–1000 bp away from the start of transcription
They can
- be downstream, upstream, or within an intron
- function in either orientation relative to the promoter
A typical enhancer:
- around 500 bp in length
- contains about 10 binding sites for several different transcription
factors

Each enhancer is responsible for a subset of the total gene expression
pattern
Enhancers increase gene promoter activity either in all tissues or in a
regulated manner
i.e. they can be
- tissue-specific or
- developmental stage-specific
They involve especially during development or in different cell types
Silencers - similar elements that repress gene activity

Three key characteristics of an enhancer element. An enhancer element can activate a promoter at a
distance (A), in either orientation (B) or when positioned upstream, downstream, or within a transcription
unit (C).

Three key characteristics of an enhancer element. An enhancer element can activate
a promoter at a distance within a transcription unit (C).

The enhancer must make contact with the transcription apparatus
When an enhancer switches a gene on, the DNA between it and the
promoter loops out
Looping
Model for
Enhancer

Binding to enhancers
increases transcriptional
levels. In the presence of basal
factors alone bound to the
promoter, low levels of
transcription occur. The binding of
activator proteins to an enhancer
element leads to an increase in
transcription beyond the basal
level.

Looping Model for Enhancer
The enhancer shown here is located downstream of the start site. To enhance transcription, the enhancer
first binds several transcription factors. Subsequently, the DNA forms a loop allowing the enhancer to make
contact with the transcription apparatus via the bound transcription factors.

Enhancers control the promotor by looping of DNA around
- the activator proteins bound at the enhancer can make contact with the
transcription apparatus via the mediator complex

Activator Proteins and the Mediator
A) The folding of the DNA allows numerous activators that are bound to enhancer sequences to approach
the transcription apparatus. B) The mediator complex allows contact of the activators and/or repressors
with the DNA polymerase.

Activator protein domains. Common motifs found in activator proteins include the helix-loop-helix, helix-turn-
helix, and zinc-fingers.

This looping mechanism allows a single enhancer to control several
genes in its vicinity
How is an enhancer prevented from activating genes further along the
chromosome, that are supposed to be under control of another, closer
enhancer?
Chromosomes are divided into regulatory neighborhoods by special
sequences known as “boundary elements,” or insulators
An enhancer is prevented from controlling a gene if an insulator sequence
lies between them on the chromosome

Insulator Sequences Restrict the Range of
Enhancer Action
A large loop of DNA is shown with an enhancer
that may interact with Gene X or Gene Y.
Insulator sequences at the base of the loop are
recognized by an IBP (insulator binding protein)
which prevents the enhancer from acting outside
of the loop.

ii. Insulator
A DNA sequence element - 300 bp to 2 kb in length
- has two distinct functions:
1. Chromatin boundary marker:
- an insulator marks the border between regions of heterochromatin and
euchromatin
2. Enhancer blocking activity:
- an insulator prevents inappropriate cross-activation or repression of
neighboring genes
- by blocking the action of enhancers and silencers

Insulators
- regions of DNA consisting of clusters of sequences that bind multiple
copies of proteins - insulator binding proteins (IBPs)
In vertebrates
- the best known is CTCF (CCCTC-binding Factor) – block enhancer
action
At least three different DNA-binding proteins recognized:
- CCCTC-binding factor (CTCF)
- Upstream stimulatory factor (USF) 1 and 2
CTCF - mediates the enhancer blocking activity
USF - bind to the insulator and recruit several chromatin-modifying enzymes

Insulators are GC-rich
CG sequences may be methylated
When the insulator element is methylated, it no longer binds the CTCF
protein and no longer functions
Ex.
The Igf2 gene (insulin-like growth factor-II) and the H19 gene are close
together and face in the same direction
Igf2 gene
The maternal copy is normally silenced
but the paternal copy is active

H19 gene
the maternal is normally active
the paternal copy is silenced
This is due to differing methylation patterns on the maternal and
paternal chromosomes
B
Methylation of Insulator Sequences and Binding

Methylation of Insulator Sequences and Binding
A) The Igf2 (insulin-like growth factor-II) gene is distant from an enhancer element. B) When the insulator is
not methylated, the CTCF protein binds to the insulator and the enhancer can only affect the H19 gene. C)
When the insulator and the H19 gene are methylated, the CTCF protein does not bind, allowing the
enhancer to activate the Igf2 gene.
C
Very few of the insulators have been described in detail
As a result, their mechanisms of action are still poorly understood

Insulators between euchromatin and heterochromatin
Eukaryotic genomes are separated into euchromatin and heterochromatin
Heterochromatin has a tendency to spread into neighboring DNA
Natural barriers to spreading are critical when active genes are nearby
1970s: Ed Lewis discovered a mutation in Drosophila affecting a chromatin
boundary
However, the general concept of boundary elements functioning as
“insulators” was not fully established until the early 1990s

Insulators function as chromatin boundary markers and have enhancer blocking activity. An
insulator (gray) marks the border between regions of heterochromatin (depicted as nucleosome-bound
DNA) and euchromatin. In erythrocytes it separates the actively transcribed β-globin genes (indicated by
the green arrow) from the inactive folate receptor gene (indicated by the red X symbol). Another insulator
prevents the inactive odorant receptor gene from cross-activation by the locus control region (LCR)
(orange) upstream of the β-globin genes, and vice versa in other cell types.

iii. Locus control regions (LCRs)/ locus activation region
DNA sequences that organize and maintain a functional domain of active
chromatin
- enhance the transcription of downstream genes
Unlike classic enhancer elements, they operate in an orientation-
dependent manner
The prototype LCR was characterized as a cluster of DNase I-
hypersensitive sites upstream of the β-globin gene cluster
Subsequently, LCRs have been shown to be present in other loci

A
B
The human β-like globin gene locus
A developmental
stage-specific
chromatin loop forms
and transcription
complexes are then
transferred from the
LCR to the
appropriate globin
gene promoter
The transfer is
facilitated by the
transcription factors
NF-E2, GATA-1, and
FOG-1

The human β-like globin gene locus. (A) Diagrammatic representation of the human β-like globin gene
locus on chromosome 11, which encodes embryonic ε-globin, the two fetal γ-globins, and the adult δ-and
β-globins. The locus control region (LCR) upstream of the ε-globin gene has five DNase I hypersensitive
(HS) sites (HS1–HS5) separated from each other by 2–4 kb. (B) Model for transcription complex
recruitment. The general transcription machinery (RNA pol II and the preinitiation complex) and other
transcriptional regulatory proteins are recruited to the LCR to form a “holocomplex.” A developmental
stage-specific chromatin loop forms and transcription complexes are then transferred from the LCR to the
appropriate globin gene promoter. The transfer is facilitated by the transcription factors NF-E2, GATA-1,
and FOG-1.

Beta-globin gene LCR is required for high-level transcription
Hemoglobin - a tetramer composed of two α-like globin polypeptides and
two β-like polypeptides plus four heme groups
α- and β-globin genes are highly regulated
In particular, the LCR of the β-like globin gene cluster provides an excellent
illustration of the complexity of regulatory regions
The β-like globin-coding regions are each 2–3 kb in size and the entire
cluster spans approximately 100 kb

The genes are expressed in erythroid cells in a tissue- and
developmental stage specific manner:
- the epsilon (ε) globin gene - activated in the embryonic stage
- the gamma (γ) globin - activated in the fetal stage
- the β-globin gene - expressed in adults
Physiological levels of expression of each of these genes can be achieved
only when they are downstream of the LCR
The DNase hypersensitive sites contain clusters of transcription factor-
binding sites

Early studies of β-globin gene expression in vivo were often inconclusive
Proper developmental regulation and high-level expression could not be
achieved coordinately in transgenic mice carrying an artificial construct
Ex. a plasmid-based β-globin gene construct
Advances in understanding LCR function came with the development of
yeast artificial chromosome (YAC) vectors

Transgenic mouse carrying a YAC construct experiments revealed that
- the LCR is required for high-level transcription of all the β-like globin
genes
- but the regulation of chromatin “loop” formation is the main mechanism
controlling developmental expression of the β-globin genes

 the LCR is a minimum requirement for globin gene expression
A transgenic mouse line constructed carrying a β-globin locus YAC that
lacked the LCR
- no detectable levels of ε-, γ-, or β-globin gene transcripts at any stage of
development

Additional in vivo mutagenesis studies
- have shown the developmental regulation of the β-like globin genes is
mediated by the regulatory elements within their individual promoters
Targeted deletions in mice
- reveal an absolute requirement of the LCR for high-level transcription of
all the β-like globin genes
- but the LCR interacts with only one gene promoter at any one time

Post-Transcriptional Regulation
RNA Editing
Similar to alternative splicing
- It can produce two proteins from the same mRNA
- the post-transcriptional modification of the base sequence of mRNA
RNA editing was first discovered in trypanosomes
Later in viruses, plants, slime molds, mammals

RNA editing in trypanosomes
Early 1980s: researchers began a study of the mitochondrial genome in
African trypanosomes
Major RNA transcripts encoding important electron transport chain
enzymes were found in mitochondria that contained nucleotides not
encoded in the DNA
The insertion of 4 uridines in the cytochrome oxidase subunit II
transcript was first reported
1987 - the addition of 34 uridines at several different sites within the 5′ end
of the cytochrome-b transcript described

These editing events were shown to:
- correct internal frameshifts
- create AUG initiation and UAG/UAA stop codons, and
- create open reading frames
The cytochrome-b mRNA was found to be edited in procyclic-form
parasites in the tsetse fly host
It is primarily unedited in bloodstream forms within the mammalian hosts

Extensive post-transcriptional editing of the cytochrome oxidase subunit III transcript in
Trypanosoma brucei. Part of the edited sequence of the cytochrome oxidase subunit III mRNA is shown.
The Us deleted in the mRNA (present as Ts in the gene) are shown in black above the sequence.

These initial reports were rapidly followed by reports of “pan-editing”
events:
- hundreds of U insertions in mitochondrial transcripts
In some mRNAs analyzed there were so many U insertions leading to
doubling of RNA length
It is now known that 12 of the 17 mtRNAs in trypanosomes undergo
RNA editing
Pre-mRNA sequences are changed by:
- the insertion of Us and
- less frequently by the deletion of Us

Mechanism for RNA editing
Guide RNAs (gRNAs)
- 50–70 nt long RNAs that specify the edited sequence
Multiple gRNAs are required to edit each pre-mRNA transcript
gRNAs are precise complementary versions of the mature mRNAs in the
edited region
The principle of RNA complementarity is used to establish the specificity of
function

The mitochondrial genome of T. brucei - “kinetoplast DNA”
- consists of:
 “maxicircles” (22 kb) – ~50 identical
 “minicircles” (1 kb) – ~10,000 heterogeneous that form a disk-shaped
structure of catenated DNA circles in situ
- The pre-edited mRNAs are encoded by the larger maxicircles
- the smaller minicircles each encode 3 - 4 gRNAs that specifiy editing
The maxicircles of different trypanosome species encode the same mRNAs
(and rRNAs) but differ in which RNAs are edited and to what extent

kDNA network structure. (A) Electron micrograph of the periphery of an isolated kDNA network from T.
avium. Loops represent interlocked minicircles (the arrowhead indicates a clear example). Bar, 500 nm. (B)
Diagrams showing the organization of minicircles. (I) Segment of an isolated network showing interlocked
minicircles in a planar array. (II) Section through a condensed network disk in vivo showing stretched-out
minicircles. The double-headed arrow indicates the thickness of the disk, which is about half the
circumference of a minicircle.

RNA editing
requires proteins
and RNAs
encoded by
mitochondrial
and nuclear
genomes within
the trypanosome

RNA editing requires proteins and RNAs encoded by mitochondrial and nuclear
genomes within the trypanosome. The catenated network of maxicircles and
minicircles that make up the kinetoplast DNA represents the mitochondrial genome. Pre-
edited mRNAs are transcribed from the maxicircles while the gRNAs are transcribed
from the minicircles. Editing complex proteins are nuclear encoded and imported into the
mitochondria (long arrow). The pre-edited mRNA is edited by uridine insertion/deletion to
generate a mature, translatable RNA.

The general mechanism of editing
Editing is catalyzed by a multiprotein complex
 the “editosome”
- has not yet been fully defined or characterized
- a complex that sediments at 20S on glycerol gradients
- contains four key enzyme activities and catalyzes in vitro editing
- editing complexes contain 7- 20 major proteins
RNA editing is restricted to the mitochondrion
- but the protein components of the editing machinery are encoded by the
nuclear DNA enzyme activities

The process of U insertion and deletion occurs by a series of enzymatic
reactions through the following steps:
1. Anchoring
2. Cleavage
3. Uridine insertion or deletion
4. Ligation
5. Repeat of editing cycle
The overall directionality of editing is from 3′ to 5′ along the mRNA

General mechanisms of insertion and deletion RNA editing
TUTase = 3′ terminal uridylyl transferase; ExoUase = Uspecific exoribonuclease

General mechanisms of insertion and deletion RNA editing. Pre-mRNAs (dark orange strands) are
edited progressively from 3′ to 5′ with each gRNA (light orange strands) specifying the editing of several
sites. Interaction between the RNAs by Watson-Crick base pairs (unbroken green lines) and GU base pairs
(blue dots) determines the sites of cleavage and the number of U nucleotides that are added or removed.
The gRNAs have a 3′ oligo(U) tail that is added post-transcriptionally. Editing occurs by a series of catalytic
steps. Cleavage of the premRNA by an endoribonuclease occurs upstream of the anchor duplex between
the pre-mRNA and its gRNA (arrow).
Us are either added to the 5′ cleavage fragment by a 3′ terminal uridylyl transferase (TUTase) or removed
by a Uspecific exoribonuclease (ExoUase), as specified by the sequence of the gRNA. The 5′ and 3′
mRNA fragments are then ligated by an RNA ligase. The process is repeated until all of the sites specified
by a gRNA are edited, resulting in complementarity (GU, AU, and GC base pairing) between the edited
mRNA and the gRNA, except at the gRNA terminals. Editing by each gRNA creates a sequence that is
complementary to the anchor region of the next gRNA to be used. This allows for the sequential use of the
multiple gRNAs that are required to edit the mRNAs in full. (Inset) Editing of the first block of Trypanosoma
brucei ATPase 6 pre-mRNA. The 3′ part of the pre-mRNA is shown with its cognate guide RNA (gA6[14]).
The gRNA specifies the insertion of 19 Us and the deletion of four Us. Inserted Us are shown in lowercase
letters and the positions of Us that have been deleted from the precursor RNA are indicated by black dots.

This extreme form of editing is apparently limited to the mitochondria of
trypanosomes
Within 5 years of the first report of editing in trypanosomes
- RNA editing events had been described in a number of organisms,
including mammals
- But, involve very different mechanisms

RNA editing in mammals
The two main classes of RNA editing enzymes in mammals
Adenosine
deaminase
Cytidine
deaminase

The two main classes of RNA editing enzymes in mammals. Adenosine deaminase (e.g. ADAR)
generates inosine from adenosine, and cytidine deaminase generates uridine from cytidine (e.g.
APOBEC1). (Inset) Ribbon model of the catalytic domain of human ADAR2. The active-site zinc atom is
represented by a magenta sphere. The N-terminal domain is colored cyan; the deamination motif region is
dark blue; and the C-terminal helical domain which, with contributions from the deamination motif, makes
the major contacts to inositol hexakisphosphate (IP6, ball and stick) is colored red.

Apolipoprotein B - plays an important role in lipid transport
- known to exist in two closely related forms
 apo-B100 - a large protein of 512 kDa synthesized by the liver
 apo-B48 - a smaller protein made by the intestine
The smaller protein is identical to the amino-terminal portion of the larger
protein
Apolipoprotein B - Example of mammalian editing

The mRNA encoding these proteins is 14.5 kb in both tissues
These two RNAs were identical with the exception of a single base at
position 6666
- a cytosine in the liver transcript and a uracil in the intestinal transcript
This change has the effect of replacing a CAA codon with a UAA stop
codon
CAA - directs the insertion of a glutamine residue
UAA stop codon - causes termination of translation of the intestine RNA
- results in the smaller protein

RNA editing of the apolipoprotein B transcript in the intestine produces an mRNA encoding the
truncated protein apo-B48.

A cytidine deaminase enzyme has been identified
- binds to the apoB mRNA at sequences adjacent to the edited site
- Converts cytosine into uracil
This enzyme is expressed in the intestine where editing of the apoB
mRNA occurs, but not in the liver
However, it is also present in other tissues: testis, ovary and spleen
- where apoB mRNA is not expressed
- indicates that this enzyme is likely to edit other transcripts expressed in
these tissues

Several other transcripts which undergo C to U editing have now been
identified
A number of different cytidine deaminase enzymes capable of carrying out
this form of editing have been characterized
An adenosine deaminase enzyme also exists in mammalian cells
Ex. in neuronal cells

Regulation of RNA stability
Cases of regulation by alterations in RNA stability
The rate of mRNA turnover plays an important role in determining its level
in the cell
A number of situations where the stability of a specific RNA species is
changed have been described
Ex. the mRNA casein turns over with a half-life of around 1 h in untreated
mammary gland cells
Following stimulation with the hormone prolactin,
- the half-life increases to over 40 h - results in increased accumulation of
casein mRNA and protein production in response to the hormone

Difference in stability of the casein mRNA in the presence (+) or absence
(–) of prolactin.

A genome-wide survey of all cellular mRNAs in yeast
- regulation at the level of mRNA stability was frequently observed for
mRNAs coding for proteins involved in rRNA synthesis and ribosome
production
 this form of regulation may be particularly frequent for genes encoding
proteins involved in the process of protein synthesis itself

Short sequences within the RNA have been identified which can
confer the pattern of stability regulation
- such regions are located in the 3′ untranslated region (3’UTR) of the
mRNA
- downstream of the stop codon
Ex.
i. the cell-cycle dependent regulation of histone H3 mRNA stability is
controlled by a 30 nucleotide sequence at the extreme 3′ end of the
molecule
ii. the destabilization of the mRNA encoding the transferrin receptor in
response to the presence of iron
- can be abolished by deletion of a 60bp within the 3’UTR

Both of these sequences have the potential to form stem-loop structures
by intra-molecular base pairing
 suggests that changes in stability might be brought about by
alterations in the folding of this region of the RNA in response to a
specific signal

Similar stem-loop structures in the human ferritin and transferrin receptor mRNAs. Note the boxed conserved
sequences in the unpaired loops and the absolute conservation of the boxed C residue, found within the stem, five
bases 5′ of the loop.

The 3’UTR is important in determining the differences in stability
observed between different RNA species
Such sequences may act either by
- promoting endonucleolytic cleavage within the RNA transcript or
- promoting loss of the poly (A) tail - opens up the RNA to exonucleolytic
attack via its free 3′ end

Binding of Proteins to mRNA Controls The Rate of Degradation
All cells contain a series of ribonucleases
- remove mRNA once it has served its function
The half-life of a typical mRNA in bacteria is 2–3 minutes
The susceptibility of mRNA to degradation depends on its secondary
structure
some mRNA molecules are inherently more stable than others

There are two main ways in which the binding of a protein might affect
mRNA stability:
i. it could directly alter susceptibility to ribonuclease attack
ii. the protein might help or hinder binding to the ribosome
- this would alter the rate of translation, and affect mRNA stability indirectly

The CsrAB regulatory system of E. coli : consists of
- an RNA-binding protein, CsrA, and
- a non-coding RNA molecule, CsrB, which acts as a dock for CsrA
The CsrA protein may either bind to those mRNA molecules that it regulates
or to CsrB RNA
The CsrAB system activates the flhDC operon by stabilizing the mRNA
CsrA protein also binds to mRNA carrying genes involved in glycogen
synthesis
The binding of CsrA hastens the decay of glg mRNA so preventing its
translation

Control of mRNA Degradation by CsrA. Stability of glg mRNA is regulated by CsrA protein. While idle, the RNA-
binding protein CsrA is bound to CsrB RNA. When CsrA protein binds to glg mRNA, the configuration of the glg mRNA
is altered to a form that is much more susceptible to degradation.

Bacterial mRNA degradation involves multiple enzymes
Bacterial mRNA is constantly degraded by a combination of
endonucleases and exonucleases
Bacterial exonucleases that act on ssRNA proceed along the nucleic acid
chain from the 3' end
Degradation of a bacterial mRNA is initiated by an endonucleolytic
attack
Several 3' ends may be generated by endonucleolytic cleavages within the
mRNA
Degradation of the released fragments of mRNA into nucleotides then
proceeds by exonucleolytic attack from the free 3 '-OH end toward the 5'
terminus

Degradation of bacterial mRNA is a two stage
process. Endonucleolytic cleavages proceed 5'-3'
behind the ribosomes. The released fragments are
degraded by exonucleases that move 3'-5'.

Cytoplasmic RNA turnover – in Eukaryotes
In the cytoplasm the mRNA may be held in a
- translationally silent state
- it may be translated and then degraded, or,
- if it contains a premature termination codon, the mRNA is rapidly
degraded by a process - nonsense-mediated mRNA decay

Alternate mRNA fates in the cytoplasm

exon–exon
junction
complexes (EJCs)

Alternate mRNA fates in the cytoplasm. Nuclear pre-mRNA processing events form mRNPs that are
exported to the cytoplasm for translation. (A) Some mRNPs are stored in the cytoplasm in a translationally
silent state. (B) Translation of wild-type mRNA. During the pioneer round of translation, ribosomes displace
exon–exon junction complexes (EJCs) from wild-type mRNAs. The absence of EJCs and positive signals
from remaining RNPs, such as the poly(A)-binding protein (PABP), ensure continued translation. Post-
translational mRNA turn-over involves general mRNA decay pathways. A common pathway is
deadenylation-independent decapping followed by 5′ → 3′ exonucleolytic digestion by Xrn1; an alternative
pathway involves deadenylation and exosome mediated 3′ → 5′ decay. (C) Model for nonsense-mediated
decay of mRNA. Failure of the ribosome to remove an EJC due to a premature termination codon (UAA) in
a nonsense mRNA leads to recruitment of Upf proteins and 5′ → 3′ “surveillance complex” scanning of the
mRNA. Interactions between a nonsense RNA and the surveillance complex result in translational
repression and rapid degradation of the RNA involving multiple decay pathways.

i. Storage of translationally silent mRNA
Ribosomes may or may not translate an mRNA immediately after its export
into the cytoplasm
Ex. in multicellular animals the oocyte (immature egg) accumulates all
the mRNAs required for early development
- no new transcription occurs until after several embryonic cell divisions
Silencing occurs through a mechanism involving shortening of their
poly(A) tails from their initial length of 200–250 adenosines to 20–40

This shortening is mediated by CPEB - a protein that binds the
cytoplasmic polyadenylation element (CPE) in the 3′ UTR of the mRNA
CPEB also interacts with Maskin - a protein that competes with eukaryotic
translation initiation factor 4G (eIF4G) for binding to eIF4E
- blocks the binding of cytoplasmic poly(A)-binding protein (PABPC)
The poly(A) tail is not essential for translation
But when RNAs lacking a poly(A) tail compete with polyadenylated RNAs
for limiting translational machinery
- the polyadenylated RNA is translated more efficiently

When oocytes are induced to complete meiosis upon fertilization,
CPEB becomes phosphorylated and
- stimulates the readdition of the poly(A) tail by cytoplasmic poly(A)
polymerases
The new poly(A) tail binds PABPC, which then recruits eIF4G to initiate
translation

General mRNA decay pathways
In most cells, the core degradation machinery attacks mRNA from its ends
Deadenylases – remove the 3′ poly(A) tail
Decapping enzymes (DCP1 and DCP2) – remove the 5′ cap
Whether a particular mRNA is degraded primarily from 3′ to 5′ or 5′ to 3′
depends on
- which set of enzymes is most active in a particular cell type and which set
is recruited most efficiently to that mRNA

Decay occurs in specialized cytoplasmic processing bodies (P bodies)
- are enriched in the decay machinery
Deadenylation-independent decapping - common degradation pathway
Because the mRNA is normally protected from degradation by the 5′ cap
- its removal by decapping enzymes causes rapid degradation of the mRNA
by a 5′ → 3′ exonuclease (XRN1)

- after the poly(A) tail has been reduced to 10 nt, the 7-methylguanosine
cap structure is hydrolyzed
Subsequently, the rest of the mRNA is degraded by the combined action of
5′ → 3′ and 3′ → 5′ exonucleases
 Deadenylation-dependent decapping pathway
- deadenylation is followed by 3′ → 5′ degradation
- requires the
 exosome - assembly of 3′ → 5′ exonucleases and
 the Ski complex - a trimeric protein complex that regulates exosome
activity

The major deadenylation-dependent decay pathways in eukaryotes. Two pathways are initiated by
deadenylation. In both, poly(A) is shortened by a poly(A) nuclease until it reaches a length of about 10 A.
Then an mRNA may be degraded by the 5′ to 3′ pathway or by the 3′ to 5′ pathway. The 5′ to 3′ pathway
involves decapping by Dcp and digestion by the Xrn1 exonuclease. The 3′ to 5′ pathway involves digestion
by the exosome complex.

Nonsense-mediated mRNA decay
Premature termination codons - arise due to gene mutation or errors in
transcription or splicing
- may encode truncated proteins that are detrimental to a cell
Nonsense mediated mRNA decay reduces the levels of these nonsense
codon-bearing mRNAs
In mammals, recognition of an mRNA with a premature termination codon
involves the assembly of protein complexes within the open reading
frame of the mRNA
These exon junction complexes (EJCs) are assembled approximately
20–24 nt upstream of each exon–exon boundary after mRNA splicing

When the first ribosome begins translating an mRNA,
- the EJCs are normally displaced as the mRNA enters the decoding center
of the ribosome
If a premature termination codon is present in the mRNA, then the
surveillance machinery is activated
UPF1, UPF2, and UPF3 proteins - are recruited to the EJC to form a
“surveillance complex”
UPF1- RNA helicase
- It associates with both translation release factors (i.e. eRF1 and eRF3)
and with UPF2 and UPF3
- Thus, it provides a link between the surveillance complex and the
translation machinery

The surveillance complexes interact with the prematurely terminating
ribosome, and promote mRNA degradation by the general mRNA decay
pathways

Two mechanisms by which a termination codon is recognized as premature. (a) In mammals, the
presence of an EJC downstream of a termination codon targets the mRNA for NMD. (b) In probably all
eukaryotes, an abnormally long 3′ UTR is recognized by the distance between the termination codon and
the poly(A)– PABP complex. In either case, the Upf1 protein binds to the terminating ribosome to trigger
decay.

A typical human or mouse gene contains 8–10 exons
These exons can be joined in different arrangements by alternative
splicing
iii. Alternative splicing
By large scale cDNA cloning and sequencing,
- >90% of the genes expressed in mammals are alternatively spliced
Thus, alternative splicing is not just the result of mistakes made by the
splicing machinery
- it is part of the gene expression program that results in multiple gene
products from a single gene locus
- The process occurs in all metazoa and is especially prevalent in
vertebrates

Modes of Alternative Splicing
The variation in mRNA sequence can take several different forms:
- Exons can be retained in an mRNA or they can be skipped
- Introns may be excised or retained
- The positions of 5’ and 3’ splice sites can be shifted to make exons
shorter or longer
- Alterations in the transcriptional start site and/or the polyadenylation
site can further contribute to the diversity of the mRNAs that are
transcribed from a single gene

Different modes of alternative splicing.

The expression of numerous cellular genes is modulated by the selection
of alternative splice sites
Thus, certain exons in one type of cell may be introns in another
Ex.
A single rat gene encodes 7 tissue-specific isoforms (splice variants) of
the muscle protein (tropomyosin)
- through the selection of alternative splice sites
A single primary transcript may undergo more than one mode of alternative
splicing
The mutually exclusive exons are normally regulated in a tissue-specific
manner

The organization of the rat -tropomyosin gene and the seven alternative splicing pathways that
give rise to cell-specific -tropomyosin isoforms.

The organization of the rat -tropomyosin gene and the seven alternative splicing pathways that
give rise to cell-specific -tropomyosin isoforms. The thin kinked lines indicate the positions occupied
by the introns before they are spliced out to form the mature mRNAs. Tissue-specific exons are indicated
together with the amino acid (aa) residues they encode: “constitutive” exons (those expressed in all
tissues) are green, those expressed only in smooth muscle (SM) are brown, those expressed only in
striated muscle (STR) are purple, and those variably expressed are yellow. Note that the smooth and
striated muscle exons encoding amino acid residues 39 to 80 are mutually exclusive; likewise, there are
alternative 3’-untranslated (UT) exons.

Complex patterns of eukaryotic mRNA splicing

Complex patterns of eukaryotic mRNA splicing. The pre-mRNA transcript of the -
tropomyosin gene is alternatively spliced in different cell types. The light green boxes
represent introns; the other colors represent exons. Polyadenylation signals are
indicated by an A. Dashed lines in the mature mRNAs indicate regions that have been
removed by splicing. TM

Alternative splicing provides a versatile means of regulating gene
expression
The splicing of most exons is constitutive
- they are always spliced or included in the final mature mRNA
However, the splicing of some exons is regulated
- they either are included or excluded from the mature mRNA

Situations where the 5′ end of the transcripts is different
- two alternative primary transcripts are produced by transcription from
different promoter elements
- then these are processed differentially
In several situations differential splicing is controlled simply by the
presence or absence of a particular exon in the primary transcript

EX. the mouse α-amylase gene
In the salivary gland
- transcription takes place from an upstream promoter
- the exon adjacent to this promoter is included in the processed RNA
- a downstream exon is omitted
In the liver
- the transcripts are initiated 2.8 kb downstream and do not contain the
upstream exon
- the processed RNA includes the downstream exon

Alternative splicing at the 5′ end of α-amylase transcripts in the liver and salivary gland. The two
alternative start sites for transcription are indicated (TATAA) together with the 5′ region of the mRNAs
produced in each tissue.

Situations where the 3′ end of the transcripts is different
After the primary transcript has been produced, it is rapidly cleaved and a
poly(A) tail is added
In many genes the process of cleavage and polyadenylation
- occurs at a different position within the primary transcript in different
tissues
- the different transcripts are then differentially spliced

Ex. genes encoding the heavy chain of the antibody molecule
The production of membrane-bound and secreted immunoglobin
molecules is controlled by the alternative splicing of different RNA
molecules differing in their 3′ ends
The longer of these two molecules contains two exons encoding the
portion of the protein that anchors it in the membrane
When this molecule is spliced, both these two exons are
included, but a region encoding the last 20 a.a. of the secreted form is
omitted

The shorter RNA - lacks the two transmembrane domain encoding exons;
but has the region specific to the secreted form
Alternative splicing of the immunoglobulin heavy chain transcript at different stages of B-cell
development. The two unspliced RNAs produced by use of the two alternative polyadenylation sites in the
gene are shown, together with the spliced mRNAs produced from them.

Calcitonin - calcium regulatory protein
The gene encoding the calcitonin protein is a small peptide of 32 a.a.
But, it also produces an RNA encoding an entirely different peptide of 36
amino acids
- named calcitonin-gene-related peptide (CGRP)
Calcitonin - produced in the thyroid gland
CGRP - produced in specific neurons within the brain and peripheral
nervous system
These two peptides are produced by alternative splicing of two distinct
transcripts differing in their 3 ends

Alternative splicing of the calcitonin/CGRP gene in brain and thyroid cells. Alternative splicing followed by
proteolytic cleavage of the protein produced in each tissue yields calcitonin in the thyroid and CGRP in the brain.

Situations where both the 5’ and 3’ ends of the differently processed
transcripts are identical
Tissue-specific splicing factors
- may also lead to transcripts identical at 5′ and 3′ ends but spliced
differently in different tissues
- This cannot be explained by differential usage of promoters or
polyadenylation sites

Ex. The troponin T gene – in skeletal muscle
The same RNA can be spliced in up to 64 different ways in different
muscle cell types
The existence of tissue-specific splicing factors acting on this gene is
indicated by the finding that
- the artificial introduction and expression of this gene in non-muscle
cells results in the complete removal of exons 4–8
- whereas in muscle cells - the correct pattern of alternative splicing
seen with the endogenous gene is reproduced faithfully

Alternative splicing of the four combinatorial
exons (4–8) and the two mutually exclusive exons (16
and 17) can result in up to 64 distinct mRNAs from
the rat troponin T gene.

Effects of alternative splicing on gene expression
The types of changes that alternative splicing confers on expressed
proteins are diverse
Particular pre-mRNAs often have multiple positions of alternative splicing
- gives rise to a family of related proteins from a single gene
- mechanism for generating protein diversity

- Splice variations may control whether a protein is:
 soluble or membrane bound
 phosphorylated by a specific kinase
 the subcellular location to which it is targeted
 whether an enzyme binds a particular allosteric effector
 the affinity with which a receptor binds a ligand

Alternative splicing can affect gene expression in the cell in at least two
ways:
i. to create structural diversity of gene products by including or omitting
some coding sequences
ii. by creating alternative reading frames for a portion of the gene
This can often modify the functional property of encoded proteins
Ex. the CaMKIIδ gene contains three alternatively spliced exons
The gene is expressed in almost all cell types and tissues in mammals
 all three alternative exons are skipped - the mRNA encodes a
cytoplasmic kinase that phosphorylates a large number of protein
substrates

 exon 14 is included - the kinase is transported to the nucleus because
exon 14 contains a nuclear localization signal
This allows the kinase to regulate transcription in the nucleus
 both exons 15 and 16 are included - the kinase is targeted to the cell
membrane
- where it can influence specific ion channel activities

Alternative splicing of the CaMKIIδ gene: different alternative exons target the kinase
to different cellular compartments.

Additional effect of alternative splicing:
 20% of mRNA variability that results from alternative splicing is within
untranslated regions
These 5′ or 3′ untranslated regions commonly contain elements that
regulate translation, stability, or localization of the mRNA
Up to 1/3rd of alternative splicing events insert premature termination
codons in the transcript
- this targets the mRNA for degradation by nonsense-mediated decay

Regulation of gene expression by alternative splicing is important in many
cellular and developmental processes:
- sex determination, apoptosis, axon guidance, cell excitation, cell
contraction
Consequently, errors in splicing regulation have been implicated in a
number of different diseases
- 15% of point mutations that cause human genetic diseases affect
splicing
Some of these mutations delete functional splice sites, thereby activating
nearby pre-existing cryptic splice sites

Many alternative splicing events have been characterized and the biological
roles of the alternatively spliced products determined
The pathway of sex determination in D. melanogaster
- the best understood example
- involves interactions between a series of genes in which alternative
splicing events distinguish males and females

The pathway starts with sex-specific splicing of sxl
Exon 3 of the sxl gene contains a termination codon that prevents
synthesis of functional protein
This exon is included in the mRNA produced in males but is skipped in
females
Thus, only females produce Sxl protein
The protein has a concentration of basic amino acids that resembles other
RNA-binding proteins
- The presence of Sxl protein changes the splicing of the transformer
(tra) gene
- involves splicing a constant 5′ site to alternative 3′ sites

One splicing pattern occurs in both males and females and results in an RNA that has
an early termination codon
The presence of Sxl protein inhibits usage of the upstream 3′ splice site by binding to the
polypyrimidine tract at its branch site
When this site is skipped, the next 3′ site is used
This generates a female-specific mRNA that encodes a protein
Thus, Sxl autoregulates the splicing of its own mRNA to ensure its
expression in females, and tra produces a protein only in females
Like Sxl, Tra protein is a splicing regulator
tra2 has a similar function in females (but is also expressed in the males)
The Tra and Tra2 proteins are SR splicing factors that act directly upon the target
transcripts

Tra and Tra2 cooperate (in females) to affect the splicing of dsx
In the dsx gene, females splice the 5′ site of intron 3 to the 3′ site of that
intron; as a result, translation terminates at the end of exon 4
Males splice the 5′ site of intron 3 directly to the 3′ site of intron 4
- exon 4 is omitted from the mRNA and allowing translation to continue
through exon 6
Result: different Dsx proteins are produced in each sex:
The male product blocks female sexual differentiation
The female product represses expression of male-specific genes

Sex determination in D. melanogaster involves a pathway in which different splicing events occur in
females

Sex determination in D. melanogaster involves a pathway in which different
splicing events occur in females.
Blockages at any stage of the pathway result in male development. Illustrated are tra
pre-mRNA splicing controlled by the Sxl protein, which blocks the use of the alternative
3′ splice site, and dsx premRNA splicing regulated by both Tra and Tra2 proteins in
conjunction with other SR proteins, which positively influence the inclusion of the
alternative exon.

Regulation of alternative splicing
Cis-acting regulatory elements - identified in exons and/or introns of
pre-mRNAs
These RNA sequence elements are bound by trans-acting proteins that
regulate splicing
 “exonic splicing enhancers” (ESEs) - regulatory elements that act to
stimulate splicing Ex. SR proteins
 “exonic splicing silencers” (ESSs) - regulatory elements that act to
repress splicing Ex. hnRNP A and B
In most systems, changes in splice site selection arise from changes in
the binding of the initial factors to the pre-mRNA, and in the assembly of the
spliceosome

Many RNA-binding proteins also affect splice-site selection through
intronic sequences
intronic splicing enhancers (ISEs) - positive cis-acting elements in introns
Intronic splicing silencers (ISSs) - negative cis-acting elements in introns

Exonic and intronic sequences can modulate splice site selection by functioning as
splicing enhancers or silencers. In general, SR proteins bind to exonic splicing
enhancers and the hnRNP proteins (e.g., the A and B families of RNA-binding proteins
[RBPs]) bind to exonic silencers. Other RBPs can function as splicing regulators by
binding to intronic splicing enhancers or silencers.

The positional effects of many splicing regulators
Ex. the Nova and Fox families of RNA-binding splicing regulators
- can enhance or suppress splice-site selection, depending on where they
bind relative to the alternative exon
Binding of both Nova and Fox to intronic sequences upstream of the
alternative exon generally results in the suppression of the exon
their binding to intronic sequences downstream of the alternative splicing
exon frequently enhances the selection of the exon

Both Nova and Fox are differentially expressed in different tissues,
particularly in the brain
Thus, tissue-specific regulation of alternative splicing can be achieved by
tissue-specific expression of trans-acting splicing regulators

The Nova and Fox families of RNA-binding proteins can promote or suppress
splice site selection in a context dependent fashion

The Nova and Fox families of RNA-binding proteins can promote or suppress
splice site selection in a context dependent fashion. Binding of Nova to exons and
flanking upstream introns inhibits the inclusion of the alternative exon, whereas Nova
binding to the downstream flanking intronic sequences promotes the inclusion of the
alternative exon. Fox binding to the upstream intronic sequence inhibits the inclusion of
the alternative exon, whereas binding of Fox to the downstream intronic sequence
promotes the inclusion of the alternative exon.

Ex. Drosophila
Genetic analysis of mutants - led to the identification of specific splicing
regulators and their downstream targets
Biochemical dissection of the regulatory mechanisms
In mammalian systems most splicing factors have been biochemically
identified
In general, the molecular mechanisms regulating tissue-specific and
developmental stage-specific control of alternative splicing remain poorly
defined

Ex. The regulation of alternative splicing of caspase-2 mRNA
- SR proteins - positive regulators of splicing
- hnRNP proteins - negative regulators of splicing
Caspase-2 has two isoforms with divergent roles in mediating apoptosis
Alternative splicing results in mRNA that either includes or lacks exon 9
Exon 9 contains a stop codon

SC35 and ASF/SF2
- promote skipping of exon 9
- results in the death-promoting (pro-apoptotic) long isoform - Casp2L
 SC35 overexpression triggers apoptosis
 hnRNP A1 overexpression is anti-apoptotic
hnRNP A1 facilitates exon 9 inclusion
- results in premature termination and
- the production of the cell survival-promoting (anti-apoptotic) short
isoform of caspase-2 - Casp2S

Examples of alternative splicing. (A) Regulation of exon 9 inclusion into the caspase-2 mRNA. An
intronic sequence element (In100) inhibits the inclusion of exon 9 into the caspase-2 mRNA. In100 is
located downstream of exon 9 and functions as a “decoy” 3′ splice site via the formation of a nonproductive
splicing complex containing the U2 snRNP. This process is modulated by binding of the polypyrimidine
tract-binding protein (PTB) downstream of U2 snRNP to In100, and is stimulated by SR proteins. Exclusion
of exon 9 leads to production of the long “pro-apoptotic” isoform of caspase-2 (Casp2L). In contrast, exon 9
inclusion is facilitated by hnRNP1. Inclusion of exon 9 causes production of a short “anti-apoptotic”
caspase-2 isoform (Casp2S), due to an open reading frame shift and a premature stop codon in exon 10.
(B) CaMKIIδ alternative splicing. The schematic drawing depicts the three alternative exons (14, 15, and
16) of the CaMKIIδ gene, and the three major isoforms containing exons 15 and 16 (δA), exon 14 (δB),
and no alternative exons (δC). Exon 14 contains a nuclear localization sequence (NLS). Immunostaining
shows the intracellular localizations of the different CaMKIIδ isoforms. The striated pattern of the δA
neuronal isoform corresponds to the T tubules of the sarcolemmal membranes. The δB cardiac isoform is
localized to the nucleus, and the δC cardiac isoform shows a largely diffuse cytoplasmic localization.
Inappropriate expression of the neuronal isoform leads to defects in heart development.

The DSCAM gene: extreme example of alternative splicing
Dscam gene – encodes downs syndrome cell adhesion molecule in
Drosophila
The Dscam gene has 95 variable exons
- can potentially generate 38,016 different protein isoforms
The gene was first described in humans and maps to a Downs syndrome
region of chromosome 21
The Dscam isoforms are a novel class of transmembrane neuronal cell adhesion
molecules that are required for the formation of neuronal connections in Drosophila
and humans

Alternative splicing of RNA transcripts of the Drosophila Dscom gene

Alternative splicing of RNA transcripts of the Drosophila Dscom gene. DSCAM proteins are axon
guidance receptors that help to direct growth c ones to their appropriate targets in the developing nervous
system. The final mRNA contains2 4 exons, four of which (denoted A , B,C ,and D) are present in the
Dscom gene as arrays of alternative exons. Each RNAc ontains1 of 12 alternativesfo r exon A (red),1o f 48
alternativesfo r exon B (green),1o f 33 alternativesfo r exonC (blue),a nd1 of 2 alternatives for exon D
(yellow). lf all possible splicing combinations are used, 38,016 different proteins could in principle be
produced from the Dscam gene. This figure shows only one of the many possible splicing patterns
(indicated by the red line and by the mature mRNA below it). Each variant Dscam protein would fold into
roughly the same structure predominantly series of extracellular immunoglobulin-like domains linked to a
membrane-spanning region (see F igure2 5-74)1b,u t the amino acid sequence of the domains would vary
according to the splicing pattern. It is suspected that this receptor diversity contributes to the formation of
complex neural circuits but the precise properties and functions of the many Dscam variants are not yet
understood.

The extracellular domain of human DSCAM is highly homologous to
Drosophila Dscam
Their intracellular domains share no obvious sequence homology
Despite these differences, human DSCAM and the Drosophila counterpart
appear to have similar biological functions in axon guidance
In contrast to the Drosophila gene, the human DSCAM gene has 30 exons
- only three different alternatively spliced transcripts have been identified

In Drosophila, four variable domains are encoded by blocks of alternative
exons
Different isoforms are expressed in specific spatial and temporal
patterns in neurons
Individual cells express around 50 different isoforms each
The array of isoforms expressed by each neuron is proposed to play an
important role in the specificity of neural wiring in the fruitfly

Extreme alternative splicing. Schematic representation of the Dscam gene, mRNA, and protein. The Dscam protein
contains both constant and variable domains. The four variable domains are encoded by alternative exons (indicated
by different colors). A transcript contains only one alternative exon from each block. The Dscam gene encodes 12
alternative exons for the Nterminal half of immunoglobulin 2 (Ig2, red), 48 alternative exons for the N-terminal half of
Ig3 (blue), and 33 alternative exons for Ig7 (dark green). There are two alternative transmembrane domains (gray).

GENES & DEVELOPMENT 34:1005–1016

Connection between defective AS and tumor heterogeneity

Connection between defective alternative splicing (AS) and tumor heterogeneity. Hypothetical
mechanism explaining the connection between defective AS and tumor heterogeneity. AS has been shown
as a mechanism regulating cell-lineage differentiation during embryogenesis. In adult tissues (on the left),
the balance between antagonistic splicing factors (i.e., heterogeneous nuclear ribo-nucleoproteins)
(hnRNPs) and SRs) contributes to the maintenance of cell differentiation. Cell adhesions and a well defined
epithelial shape (bottom left) characterize epithelial cells (light pink). In a physiological context, they receive
oxygen and nutrients by blood vessels (red) and interact with surrounding stromal cells (orange). In a
pathological context, aberrant extracellular signals or stochastic mutations dramatically affect the balance
in antagonistic splicing factors (on the right) leading to tumor heterogeneity. Differentiated (light pink) and
stem-like (orange) cancer cells coexist in the same tumor bulk. Their interaction with surrounding stromal
cells may sustain neo-angiogenesis and activate invasive programs at later stages (bottom right).

Common splicing regulatory RNA-binding proteins

Common splicing regulatory RNA-binding proteins.
Domain schematics for each factor are displayed. (RRM) RNA recognition motif; (psRRM) pseudo-RRM;
(RS) arginine/serinerich; (Zn) zinc finger; (Gly) glycine-rich region; (P) proline-rich region; (RGG)
arginine/glycine/glycine repeat region; (RS) arginine/ serine-rich; (KH) K homology domain. Binding
preferences for each factor are specified for SRSF1 (Ray et al. 2013), SRSF2 (Kim et al. 2015; Zhang et
al. 2015), SRSF7 (Ray et al. 2013), hnRNP A1 (Ray et al. 2013), PTB (Xue et al. 2009), hnRNP L (Hui et
al. 2005), and hnRNP K (Klimek-Tomczak et al. 2004).

Int. J. Mol. Sci. 2020, 21, 6995

Simplified model of the human IGF1 gene structure (A), featuring main mRNA isoforms (variants)
generated by alternative splicing and encoded precursor peptides (B), with the three-dimensional structure
of IGF1 protein determined by X-ray crystallography, based on PDB no. 1IMX (C). The human IGF1 gene
is composed of 6 major exons and a newly discovered exon 0, upstream of exon 1. Splicing and exons in
the human IGF1 gene generate distinct transcripts that vary in the 50 and 30 ends though the mature IGF1
protein is invariant. Transcription starts from one of the two promoters (P1 and P2) located in exon 1 and 2,
respectively. Exons 1 and 2 are alternatively utilized and comprise IGF1 class I and II, respectively. Exons
3 and 4 are expressed in all known isoforms. Exon 5 is absent in isoform A (class I/II), but it forms isoforms
B and C (class I/II). Transcripts containing exon 4 spliced directly to exon 6 are also referred to as IGF1Ea,
those containing exon 5 spliced to exon 4 (and lacking exon 6) are referred to as IGF1Eb (unique to
humans). The IGF1Ec splice variant in humans is an exon 4–5–6 variant. All peptide products derived from
pro-IGF1 are shown.

In vivo expression of different IGF1 isoforms (mRNA, protein) in selected human cancers

Trans-splicing
In rare cases, an exon from one pre-mRNA can join to an exon from
another pre-mRNA  trans-splicing
Except for its intermolecular nature, trans-splicing exactly parallels the two
steps of cis-splicing in group II introns and pre-mRNA spliceosomal introns
Trans-splicing is rare
But is now known to occur in some diverse organisms: flatworms, the protist
Euglena gracilis, plant organelles, nematodes, and Drosophila
At present there is no evidence that the low level of trans-splicing observed
in mammalian cells leads to the production of proteins with essential
functions

Genome Biol. Evol. 8(3):562–577; 2016

Schematic diagram of different types of pre-RNA splicing events. (A) Cis-splicing. After excision of
introns, exons of the same pre-mRNA are joined together to form a linear molecule. (B) Intergenic trans-
splicing. Transcripts from different genes or even different chromosomes could be spliced and generate a
non-linear chimeric molecule.

(C) Intragenic trans-splicing. Boxes with vertical line represent exons transcribed from the other strand. In
the same gene, splicing reaction occurs between two identical transcripts, alternatively, transcripts from
different strands leading to exon-duplication and sense–antisense fusion. (D) SL trans-splicing. Red boxes
represent structural genes, while T represents for the TMG cap on Spliced-leader (SL) mini-exon. SL exon
produced from tandem repeated SL gene cluster, splicing reaction occurs between SL exon and distinct
structural genes of a ploycistronic pre-mRNA to generate an array of mature “capped” transcripts.

Schematic representation of proposed models of trans-splicing mechanisms. (A) tRNA-mediated trans-
splicing model. Pre-tRNA halve adjacent to pre-mRNA context narrowing two associated molecules through
complementary sequences, then the hybrid molecule is cleaved precisely at the sites of the tRNA intron by tRNA
splicing endonuclease. (B) Transcriptional slippage model. Gray boxes represent pairing of SHSs. A pre-RNA is
transcribed from Gene 1 and then misaligns to the DNA template of gene 2 via the SHSs. Transcription machinery
keeps on moving on the strand of gene 2, after removal of introns, resulting in the chimeric molecule.

(C) Special case of transcriptional slippage model. Both partner genes share a forward direction repeat
sequence in the junction site of chimeric RNA. (D) Spliceosome mediated trans-splicing model. Like
canonical cis-splicing, pre-RNA 1 and pre-RNA 2 is precisely spliced at the 5 - and 3 -splicing site and
ligated as a non-linear chimeric molecule.

Transcription II_Adv Mol Biol_MSc_2023-24.pdf

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