This document discusses transcription in prokaryotes and eukaryotes. In prokaryotes, transcription is carried out by RNA polymerase, which associates with sigma factors to form holoenzymes that recognize specific promoter sequences. The process involves initiation at promoters, elongation, and termination. In eukaryotes, there are three nuclear RNA polymerases that transcribe different genes. RNA polymerase II transcribes protein-coding genes using promoters that often contain TATA boxes and other recognition elements.
1. Part II The Transfer of
Genetic Information
Chapter 3 Transcription
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering
College of Life Science and Technology, Xinjiang University
Li yi-jie
jaylixj@hotmail.com
2.
3. Content:
Some basic conception
Transcription in the Prokaryotic Nucleus
Transcription in the Eukaryotic Nucleus
mRNA Differences between Prokaryotes and
eukaryotes
Production of mature mRNA in Eukaryotes
Transcription in No Code Protein Genes
4. Gene
A region of a DNA molecule containing a sequence of
bases that is transcribed into a functional product.
Several regions are responsible for the proper function
of a gene.
Regulatory region-sequence of bases that control the
initiation of transcription.
Coding region- sequence of bases that are read into a
functional molecule (RNA or protein).
Termination region-sequence of nucleotides that stops
transcription.
5. When a protein is needed by a cell, the genetic code for that protein must
be read from the DNA and processed.
A two step process:
1. Transcription = synthesis of a single-stranded RNA molecule
using the DNA template (1 strand of DNA is transcribed).
In Eukaryotes, transcription takes place in the nucleus.
2. Translation = conversion of a messenger RNA sequence into
the amino acid sequence of a polypeptide (i.e., protein
synthesis)
Translation takes place on ribosomes in the cytosol, or on rough
ER
Both processes generally occur throughout the cell cycle.
6. Template strand: The 3’ to 5’ strand of DNA in a
coding region that serves as the template for RNA
synthesis. The resulting RNA is a complement of
the template strand.
Nontemplate strand: The 5’ to 3’ strand of DNA in
a coding region that has no function in producing
the RNA. It has the same sequence as the
initially transcribed RNA, except that Thymine in
the DNA is Uracil in the RNA.
7. Sense/antisense
+/-
Nontranscribed/transcribed
Nontemplate/template
coding/template
Conventions for describing the strands
8. Both strands encode genes
Either strand of a DNA molecule may be used
as a template, but transcription always reads
the 3’ to 5’ strand relative to the direction of
RNA polymerase movement.
9. mRNA--(messenger) intermediate molecules used for
transfer of information from DNA to protein.
rRNA--(ribosomal) functional RNA molecules that are
components of the ribosome.
tRNA--(transfer) functional RNA molecules that serve
as adapters in translation.
snRNA--(small nuclear) functional RNA molecules that
are involved in the removal of introns from pre-
mRNAs.
scRNA--(small cytoplasmic) functional RNA molecules
that are involved in protein traficking with the
cytoplasm.
Various other functional RNAs
Five different types of RNA:
10. Unlike DNA replication, we will only
want to copy specific sequences at
specific times in specific cell types
We will need specific start and stop
signals
Only one strand will be copied
11. How is an RNA strand synthesized?
1. Regulated by gene regulatory elements within each gene.
2. DNA unwinds next to a gene.
3. RNA is transcribed 5’ to 3’ from the template (3’ to 5’).
4. Similar to DNA synthesis, except:
RNA polymerase
No primer
No proofreading
NTPs instead of dNTPs (no deoxy-)
Adds Uracil (U) instead of thymine (T)
14. There is only one RNA polymerase in E.
coli
Responsible for the synthesis of all types
of RNA
We will first look at the core enzyme and
then at the holoenzyme
Ds-DNA dependentdependent
E. coli RNA polymerase
15. Capable of polymerization but not
initiation
RNA polymerase core
enzyme
16. Core enzyme + sigma factor
The sigma factor is a separate protein
required for initiation
There are many different sigma factors
Different sigma factors allow for the
expression of different genes
RNA polymerase holoenzyme
α2ββ’σ α2ββ’ + σ
holoenzyme core polymerase sigma factor
17. The roles of each subunitThe roles of each subunit
18. Functions of other subunits:
1. α - binds the UP element found upstream of very strong
promoters (rRNA), and activators
2. β - active site of Pol, also binds nascent RNA, RNA-
DNA hybrid, and DS DNA in front of the bubble
3. β’ – also binds nascent RNA, RNA-DNA hybrid, and DS
DNA in front of the bubble
4. σ -factor confers specificity to the polymerases, directs
polymerase to initiate at specific sites, called promoters.
20. Loose Loose
Looser Tight
promoter sites
non-promoter sites
non-promoter sites
promoter sites
Pol Core has non-specific affinity for DNA
that is reduced by σ.
21. The function of sigma factor
• the sigma subunit of RNA polymerase is an “initiation factor”
• there are several different sigma factors in E. coli that are
specific for different sets of genes
• sigma factor functions to ensure that RNA polymerase binds
stably to DNA only at promoters
• sigma destablizes nonspecific binding to non-promoter DNA
• sigma stabilizes specific binding to promoter DNA
• this accelerates the search for promoter DNA
Ka (M-1
)
Any DNA Promoter DNA
(nonspecific) (specific)
Core 2 X 1011
Holo 1 X 107
1013
to 1015
• promoters vary in “strength” by ~two orders of magnitude
23. The fidelity of RNAThe fidelity of RNA
polymerasepolymerase
No proofreading nuclease activities
1 error in 105
bp
About 105
lower than replication
27. Recognition sequences in theRecognition sequences in the
promoterpromoter
The Primbnow box which includes
-10 consensus sequence: TATAAT
-35 consensus sequence: TTGACA
28. How does RNA polymerase findHow does RNA polymerase find
the promoter?the promoter?
RNA polymerase scans DNA linearly
Slides along DNA without opening strands
Detects sequences of -10 and -35 in the major
groove
Sigma factor allows RNA polymerase to bind
to promoter
29. The first events at the promoterThe first events at the promoter
RNA polymerase binds to the -35 region
This is called a closed complex because the
DNA is still base-paired
31. 1. Banding
2. Initiation
3. Elongation
4. Termination
Occur in both prokaryotes
and eukaryotes.
Elongation is conserved in
prokaryotes and eukaryotes.
Initiation and termination
proceed differently.
33. E. coli model:
Each gene has three regions:
1. 5’ Promoter, attracts RNA polymerase
e.g., -10 bp 5’-TATAAT-3’
e.g., -35 bp 5’-TTGACA-3’
2. Transcribed sequence, or RNA coding sequence
3. 3’ Terminator, signals the stop point
Minus numbers represent bases upstream of mRNA start point, +1 is the first base in
the RNA transcript.
34. 1. RNA polymerase combines with sigma factor (a polypeptide) to create
RNA polymerase holoenzyme
Recognizes promoters and initiates transcription.
Sigma factor required for efficient binding and transcription.
Different sigma factors recognize different promoter sequences.
2. RNA polymerase holoenzyme binds promoters and untwists DNA
e.g., binds loosely to the -35 promoter (DNA is d.s.)
e.g., binds tightly to the -10 promoter and untwists
3. Different types and levels of sigma factors influence the level and
dynamics of gene expression (how much and efficiency).
36. Step 3-Elongation…
--RNA polymerization within a moving
bubble (about 18 bases) of melted helix.
Ribonucleoside triphosphates serve as the precursors
for synthesizing RNA.
Except for the first, all ribonucleotides are added to an
existing 3’ hydroxyl group of an existing nucleotide.
– Two external phosphates are lost as the internal forms a
phosphodiester bond with the hydroxyl group at the 3’ carbon
of the ribose.
Due to this chemistry, an RNA molecule grows at its 3’
end, thus is synthesized in a 5’ to 3’ direction.
The sequence of the RNA is dictated by the template
strand of DNA, which is organized 3’ to 5’ relative to
the direction of transcription.
37.
38. 1. After 8-9 bp of RNA synthesis occurs, sigma factor
is released and recycled for other reactions.
2. RNA polymerase completes the transcription at 30-
50 bp/second.
3. DNA untwists rapidly, and re-anneals behind the
enzyme.
4. Part of the new RNA strand is hybrid DNA-RNA,
but most RNA is displaced as the helix reforms.
39. A. After ~10 nucleotides have been added, 5’ end
ribonucleotide unpairs from template.
B. The σ subunit dissociates from core.
C. The size of RNA-DNA hybrid maintained during
elongation.
D. Sigma recycles to new polymerase molecules.
40. Step 4-Termination…
--stopping the synthesis of an RNA
Different mechanisms of termination
Prokaryotes
– rho-independent termination: formation of a hairpin
structure
– rho-dependent termination: external protein disrupts
transcription
Eukaryotes
– cleavage of the RNA by an external protein
41. Two types of terminator sequences occur in prokaryotes:
1. Type I (ρ-independent)
Palindromic, inverse repeat forms a hairpin loop and is believed to
destabilize the DNA-RNA hybrid.
2. Type II (ρ-dependent)
Involves ρ factor proteins, believed to break the hydrogen bonds
between the template DNA and RNA.
47. Rho dependentRho dependent
Rho factor has an ATP-dependent RNA-
DNA helicase activity
Still have a hairpin but no poly(U) stretch
In both cases, the signal to terminate
transcription is present in the newly
transcribed RNA, not in the DNA
49. Rho-Dependent:
“Rho” is a bacterial Termination
Factor
It acts as a hexamer of 46 kD subunits
- binds a specific 72 base sequence
of ssRNA
It then hydrolyses ATP and eventually
disrupts pairing between the nascent
strand & template
54. Prokaryotes possess only one type of RNA polymerase
transcribes mRNAs, tRNAs, and rRNAs
Transcription is more complicated in eukaryotes
Eukaryotes possess three RNA polymerases:
1. RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S
2. RNA polymerase II, transcribes mRNAs and some snRNAs
3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs
*S values of rRNAs refer to molecular size, as determined in a sucrose
gradient (review box 5.1)
55. Polymerase Genes transcribed Subcellular
localization
sensitivity to
α amanitin
RNA
polymerase I
rRNA genes
this is actually the bulk
of cellular transcription
nucleolus insensitive
RNA
polymerase II
protein encoding genes
are transcribed to
produce mRNA
nucleoplasm
(everything
but the
nucleolus)
usually very
ensitive
RNA
polymerase
III
tRNAs, 5S RNA and other
small nuclear RNAs
nucleoplasm sensitivity
depends on
species
Summary of RNAP roles and location
56. Promoters for the 3 nuclearPromoters for the 3 nuclear
RNA polymerases (nRNAPs)RNA polymerases (nRNAPs)
Order of lecture topics:
1. Class II promoters (for nRNAP II)
2. Class I promoters (for RNAP I)
3. Class III promoters (for RNAP III)
4. Enhancers and Silencers
57. RNAP II Promoters (a.k.a. Class II)
Class-II promoters usually have 4 components:
1. Upstream element
2. TATA Box (at approx. –25)
3. Initiation region
4. Downstream element
Many class II promoters lack 3 and 4.
1. 2. 3. 4.
58. TATA Box of Class II PromotersTATA Box of Class II Promoters
TATA box = TATAAAA
Defines where transcription starts.
Also required for efficient transcription for
some promoters.
Some class II promoters (e.g., for
housekeeping genes or some
developmentally regulated genes (e.g.,
homeotic)) don’t have a TATA box.
59. Transcription starts at a purine ~25-30 bp from the TATA box.Transcription starts at a purine ~25-30 bp from the TATA box.
SV40 early
promoter
analyzed
in vivo.
Normal promoter.
60. Upstream Elements of Class IIUpstream Elements of Class II
Can be several of these
Two that are found in many class II promoters:
1. GC boxes (GGGCGG and CCGCCCC)
– Stimulate transcription in either orientation
– May be multiple copies
– Must be close to TATA box (different from enhancers)
– Bind the Sp1 factor
2. CCAAT box
– Stimulates transcription
– Binds CCAAT-binding transcription factor (CTF) or
CCAAT/enhancer-binding protein (C/EBP)
61.
62. Class I Promoters (for nRNAP I)Class I Promoters (for nRNAP I)
Sequences less conserved than Class II
Usually 2 parts:
– UCE : upstream control element , -150 to -100
in human rRNA
– Core: from - 45 to +20
Spacing between elements also important
-150 -100 -50 +20
UCE Core
63. Class III promoters (for nRNAP III)Class III promoters (for nRNAP III)
2 types:
1. Internal promoters
- 5S rRNA (Box A, Intermediate element, Box C)
- tRNA (Box A, B)
2. Class II – like promoters
- contain TATA box
- 7SL gene, promoter is upstream of coding region
64. Enhancers and SilencersEnhancers and Silencers
Enhancers stimulate transcription, silencers
inhibit.
Both are orientation independent.
– Flip 180 degrees, no effect
Both are position independent.
– Can work at a distance from promoter
– Enhancers have been found all over
Bind regulated transcription factors.
65. Transcription Factors for Class IITranscription Factors for Class II
promoters (RNAP II)promoters (RNAP II)
• Basal factors
Required for initiation at nearly all promoters; determine site of
initiation; interact with TATA box.
• Upstream factors
DNA binding proteins that recognize consensus elements upstream of
TATA box. Ubiquitous. Increase efficiency of initiation. Interact with
proximal promoter elements (e.g., CCAAT box).
• Inducible (regulated) factors
Work like upstream factors but are regulatory. Made or active only at
specific times or in specific tissues. Interact with enhancers or
silencers.
66. Transcription of protein-coding genes by RNA polymerase II
Basal transcription factors (TFs) also are required by RNA
polymerases.
TFs are proteins, assembled on basal promoter elements
Each TF works with only one kind of RNA polymerase (required by all
3 RNA polymerases).
Numbered (i.e., named) to match their RNA polymerase.
e.g., TFIID, TFIIB, TFIIF, TFIIE, TFIIH
Binding of TFs and RNA polymerase occurs in a set order in protein
coding genes.
Complete complex (RNA polymerase + TFs) is called a pre-initiation
complex (PIC).
68. Binding of Activator Factors
Finally, high-level transcription is induced by the binding of activator
factors to DNA sequences called enhancers.
Single or multiple copies in either orientation
Usually located upstream
Can be several kb from the gene
Silencer elements and repressor factors also exist
70. Prokaryotes
1. mRNA transcript is mature, and used directly for translation
without modification.
2. Since prokaryotes lack a nucleus, mRNA also is translated on
ribosomes before is is transcribed completely (i.e.,
transcription and translation are coupled).
3. Prokaryote mRNAs are polycistronic, they contain amino
acid coding information for more than one gene.
71. Eukaryotes
1. mRNA transcript is not mature (pre-mRNA) and must be
modified by processing.
2. Transcription and translation are not coupled (mRNA
must first be exported to the cytoplasm before translation
occurs).
3. Eukaryote mRNAs are monocistronic, they contain amino
acid for just one gene.
76. 1. Gene is transcribed by RNA polymerase II.
2. Introns are removed and exons are spliced.
77. Post-transcriptional processing of
mRNA in eukaryotes
Initial transcript is known as the pre-mRNA, and
before being exported from the nucleus processing
must occur. Final processed mRNA is exported to the
cytoplasm.
RNA processing:
1. Addition of 7-methylguanosine cap to the 5’ end by
guanyltransferase.
2. Addition of poly-Adenosine tail at the 3’ end by
poly(A) polymerase.
3. Removal of introns by the spliceosome
78. RNA Capping
1. Post-transcriptional (i.e., G not encoded)
2. Involves adding a 7Me
Guanosine Nt to the first (RNA) Nt in
an unusual way, and often methylation of the first few nt of
the RNA.
3. Occurs before the pre-mRNA is 30 nt long
4. The 5’-end of the mRNA is capped 5’ to 5’ with a guanine
nucleotide.
– Results in the addition of two methyl (CH3) groups.
– Essential for the ribosome to bind to the 5’ end of the
mRNA.
81. Cap Functions
Cap provides:
1. Protection from some
ribonucleases
2. Enhanced translation
3. Enhanced transport from nucleus
4. Enhances splicing of first intron for
some mRNAs
82.
83. Post-transcriptional Processes II:
Pre-mRNA Polyadenylation
Most cytoplasmic mRNAs have a polyA tail (3’ end) of
50-250 Adenylates
– a notable exception is histone mRNAs
Added post-transcriptionally by an enzyme, polyA
polymerase(s)
Turns over (recycles) in cytoplasm
84. Functions oF the PolyA
tAil
1. Promotes mRNA stability
- Deadenylation (shortening of the polyA
tail) can trigger rapid degradation of the
mRNA
2. Enhances translation
- promotes recruitment by ribosomes
- bound by a polyA-binding protein in
the cytoplasm called PAB1
- synergistic stimulation with Cap!
85. Methods: Firefly luciferase mRNAs (with 5’ and 3’ UTRs from a plant
gene) were electroporated into protoplasts. At intervals, the amount of
luciferase mRNA was checked (to determine half-life), and the
amount of luciferase activity (which reflects the amount of translation
product).
86. Overview of Polyadenylation
Mechanism
1. Transcription extends
beyond mRNA end
2. Transcript is cut at 3’ end
of what will become the
mRNA (in green)
3. PolyA Polymerase adds
~250 As to 3’ end
4. “Extra” RNA (in red)
degraded
87. Evidence for Transcription Past
the End of a Cellular mRNA
Nuclear run-on transcription assay using Friend
erythroleukemic cells treated with DMSO
(stimulates globin gene transcription)
– Newly synthesized RNAs are hybridized to DNA
regions that span the globin gene, including
regions downstream
– The amount of newly synthesized RNA
complementary to each gene region is thus
quantified
89. Transcription beyond the polyA site.
After run-on transcription (in the presence of 32
P-UTP) with nuclei from
Friend Erythroleukemic cells, the labeled RNA was hybridized to DNA
fragments A-F that span the globin gene. The relative molarities of newly
synthesized RNA that hybridized to each fragment are given. s.d. is the
standard deviation.
Notice that there is just as much RNA transcribed from
downstream of the gene (E) as there is within the gene (C).
90. Polyadenylation (PolyA) Signals
AAUAAA in mammals and plants
Located ~20-30 bp from the polyA site
– Other hexamers less efficient but are used
Mutagenesis and in vivo expression studies
reveal 2 other motifs downstream of AAUAAA
that are important:
1. GU-rich stretch
2. U-rich stretch
91. What if there are multiple possible
signals?
Competition experiment:
1. The synthetic polyadenylation site (SPA) below
was inserted downstream of a normal one in a
globin gene
Fig. 15.21
GU-rich U-rich
92. 2. The modified globin gene was introduced into
HeLa cells, and the 3’ end of the mRNA analyzed
by S1 mapping
Result:
SPA was mainly used for polyAdenylation
Conclusion
Stronger set of signals in the SPA, which
“outcompeted” the native globin polyA signal (it
lacks the U-rich motif)
93. Polyadenylation: The Proteins
Several proteins are required in mammals for cleavage and
polyadenylation.
Proteins required for efficient cleavage of pre-mRNA:
1. CPSF (cleavage and polyadenylation specificity
factor), binds the AAAUAA
2. CstF (cleavage stimulation factor) binds to the G/U rich
region cooperatively with CPSF
3. CFI and CFII (cleavage factors I and II), RNA-binding
proteins
4. PAP (poly A polymerase)
5. nRNAP II (the CTD of the very large RPB1 subunit)
stimulates cleavage.
95. Polyadenylation: Mechanism
Occurs in 2 phases
–Phase 1: requires AAUAAA and ~8 nt
downstream (3’)
–Phase 2 : Once ~10 As are added,
further adenylation does not require
the AAUAAA
96. 2 Phases to Polyadenylation
Fig. 15.28
substrates:
1. 58-nt RNA from SV40
that ends with AAUAAA plus
8 nt
2. same RNA as in 1 plus
A40 tail
3. same RNA as in 1 plus a
40-nt 3’ tag from a vector
sequence
The series with an X contain
a mutated AAUAAA
(AAGAAA).
Hela cell nuclear extract incubated with various radiolabeled RNA
substrates, which were then separated by gel electrophoresis.
Conclusion: the AAUAAA not needed for phase 2, only phase 1.
97. Proteins Required for
Polyadenylation
Phase I:
1. CPSF
2. PolyA polymerase
Phase II:
1. PolyA polymerase
2. PolyA Binding Protein II (PAB II)
- PAB II binds to short A-tail
- Helps PAP synthesize long tails
98. Nuclear PolyA Polymerase (long
form = PAPII)
RBD - RNA binding domain
NLS - nuclear localization signal
PM - polymerase module
S/T- serine/threonine rich regions (yellow)
100. RNA Splicing
RNA splicing is the
removal of
intervening
sequences (IVS)
that interrupt the
coding region of a
gene
Excision of the IVS
(intron) is
accompanied by
the precise ligation
of the coding
regions (exons)
101.
102. Introns and exons:
Eukaryote pre-mRNAs often have intervening introns that must be
removed during RNA processing (as do some viruses).
intron = non-coding DNA sequences between exons in a gene.
exon = expressed DNA sequences in a gene, code for amino acids.
1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)
103. mRNA splicing of exons and removal of introns:
1. Introns typically begin with a 5’-GT(U) and end with AG-3’.
2. Cleavage occurs first at the 5’ end of intron 1 (between 2 exons).
3. The now free G joins with an A at a specific branch point sequence in
the middle of the intron, using a 2’ to 5’ phosphodiester bond.
Intron forms a lariat-shaped structure.
4. Lariat is excised, and the exons are joined to form a spliced mRNA.
5. Splicing is mediated by splicosomes, complexes of small nuclear RNAs
(snRNAs) and proteins, that cleave the intron at the 3’ end and join the
exons.
6. Introns are degraded by the cell.
104. Intron Classes & Distribution
1. Group I - common in organelles, nuclear
rRNA genes of lower eukaryotes, a few
prokaryotes and some phage genes
2. Group II - common in organelles, also in
some prokaryotes
3. Nuclear mRNA (NmRNA) - ubiquitous in
eucaryotes
4. Nuclear tRNA- some eucaryotes
105. Relationships of the 4 Intron
Classes
1. Each class has a distinctive structure
2. The chemistry of the Groups I, II and
NmRNA reactions are similar – i.e,
transesterification reactions
3. The splicing pathway for Group II and
nuclear mRNA introns are similar
4. Splicing of Groups I, II and possibly
NmRNA introns are RNA-catalyzed
106. Self-Splicing Introns
1. Some Group I and Group II introns can
self-splice in vitro in the absence of
proteins (or other RNAs), i.e. they are
ribozymes.
2. Each group has a distinctive, semi-
conserved secondary structure.
3. Both groups require Mg2+
to fold into a
catalytically active ribozyme.
4. Group I introns also require a guanosine
nucleotide in the first step.
110. 1. A snurp contains a small, nuclear U-rich RNA
(snRNAs = U1, U2, U4, U5 or U6), and proteins
(at least 7).
2. The snRNAs base-pair with the pre-mRNA (U1,
U2, U5, U6), and some with each other (U4-U6
pair in snurps, and U2-U6 pair in spliceosome).
3. Lupus patients have antibodies to snurps.
Spliceosomes contain Snurps
(a.k.a., snRNPs or small nuclear
ribonucleoprotein particles)
111. 1. U1 base-pairs with the 5’ splice-site
2. U2 binds/pairs with the branch point; also pairs
with U6 in the assembled spliceosome
3. U4 pairs with U6 in SnRNPs, but unpairs during
spliceosome assembly
4. U5 interacts with both exons (only 1-2 nt adjacent to
intron); helps bring exons together
5. U6 displaces U1 at the 5’ splice-site (pairs with nt in
the intron); it also pairs with U2 in the catalytic center
of the spliceosome
Roles of snRNAs/Snurps
116. RNA Editing
Definition: any process, other than splicing,
that results in a change in the sequence of
a RNA transcript such that it differs from
the sequence of the DNA template
1. Adds or deletes nucleotides from a pre-RNA, or
chemically alters the bases, so the mRNA bases do not
match the DNA sequence.
2. Results in the substitution, addition, or deletion of
amino acids (relative to the DNA template).
3. Generally cell or tissue specific.
117. Examples: protozoa, slime molds, plant
organelles, mammals
Discovered in trypanosome mitochondria
Also common in plant mitochondria
Also occurs in a few chloroplast genes of
higher plants, and at least a few nuclear
genes in mammals
118. Discovery of RNA Editing in
Trypanosome Mitochondria
Unusual Mitos. called Kinetoplasts
DNA:
– Maxicircles (22 kb in T. brucei), contains most of the
genes
– Minicircles (1-3 kb), heterogenous
Sequencing of genomic Mt DNA (Maxicircles)
revealed apparent pseudogenes:
– Full of Stop codons
– Deletions of important amino acids
120. Where were the real functional genes?
Investigators generated cDNA clones to some of
the kinetoplast mRNAs and sequenced them
Sequences were partially complementary to
pseudogenes on maxicircle DNA
e.g., cytochrome oxidase
subunit III
– the COXIII DNA sequence above is missing 4 Us
found in the mRNA
Called this “Editing” because it produced functional mRNAs
and proteins from pseudogenes
122. Editing Mechanism
Post-transcriptional
Guide RNAs (gRNAs) direct editing
– gRNAs are small and complementary to portions of
the edited mRNA
– Base-pairing of gRNA with unedited RNA gives
mismatches, which are recognized by the editing
machinery
– Machinery includes an endonuclease, a Terminal
UridylylTransferase (TUTase), and a RNA ligase
Editing is directional, from 3’ to 5’
123. Guide RNAs Direct Editing in
Trypanosomes.
Editing is from 3’ to 5’ along an unedited RNA.
125. Other Systems with RNA Editing
Land plant (C U) and Physarum (slime
mold) mitochondria (nt insertions)
Chloroplasts of angiosperms (C U)
A few nuclear genes in mammals
– Apolipoprotein B (C U)
– Glutamate receptor [A I (inosine)]
Hepatitus delta virus (A I)
Paramyxovirus (G insertions)
126. Post-Transcriptional Gene
Silencing (PTGS)
Also called RNA interference or RNAi.
Process results in down-regulation of a gene
at the RNA level (i.e., after transcription).
There is also gene silencing at the
transcriptional level (TGS).
Examples: transposons, retroviral
genes, heterochromatin
127. Discovery of PTGS
First discovered in plants (1990)
Introduction of transgenes homologous to
endogenous genes often resulted in
plants with both genes suppressed!
Called Co-suppression
Resulted in degradation of the endogenous
and the transgene mRNA
128. Discovery of PTGS (cont.)
Involved attempts to manipulate pigment
synthesis genes in petunia
Genes were enzymes of the flavonoid/
anthocyanins pathway:
–CHS - chalcone synthase
–DFR - dihydroflavonol reductase
When these genes were introduced into petunia
using a strong viral promoter, mRNA levels
dropped and so did pigment levels in many
transgenics
130. DFR construct introduced into petunia
CaMV - 35S promoter from Cauliflower
Mosaic Virus
DFR cDNA – cDNA copy of the DFR
mRNA (intronless DFR gene)
T Nos - 3’ processing signal from the
Nopaline synthase gene
Flowers from 3 different Transgenic petunia plants that carry copies of the
chimeric DFR gene above. These flowers showed reduced DFR mRNA levels
(lower than wild-type) in the non-pigmented areas.
131. Antisense Technology
Antisense technology has been used for ~20
years
Based on introducing an antisense gene (or
antisense RNA) into cells or organisms to
try to block translation of the sense
mRNA.
Alternative to gene knock-outs, which are
very difficult to do in higher plants and
animals.
132. The “antisense effect” was probably due to RNAi
rather than inhibiting translation
RNAi discovered in C. elegans (the first animal)
while attempting to use antisense RNA in vivo
–“Sense” control RNAs also produced suppression
of target gene!
–turned out that the sense (and antisense) RNAs
were contaminated by small amounts of dsRNA
–dsRNA was the suppressing agent
133. Double-stranded RNA (dsRNA) induced interference of
the Mex-3 mRNA in the nematode C. elegans.
Antisense RNA (c) or
dsRNA (d) for the mex-3
(mRNA) was injected into
C. elegans ovaries, and
then mex-3 mRNA was
detected in embryos by in
situ hybridization with a
mex-3 probe.
(a) control embryo
(b) control embryo hyb.
with mex-3 probe
Conclusion: dsRNA reduced mex-3 mRNA better than antisense mRNA. Also, the
suppression signal moves from cell to cell.
Fig. 16.37
134. PTGS occurs in wide variety of in
Eukaryotes
called RNA interference or RNAi in:
– C. elegans (nematode)
– Drosophila
– Mammalian cells
called “quelling” in Neurospora
Not detected (yet) in Yeast!
135. Mechanism of RNAi
Some facts and findings:
1. Cells (plants and animals) undergoing RNAi contain small
RNAs (~25 nt) that seem to result from degradation of the
target mRNA.
2. A nuclease was purified from Drosophila embryos
undergoing RNAi that digested the target mRNA.
• The nuclease contained associated small RNAs (both
sense and antisense)
• Degradation of the small RNAs with micrococcal
nuclease prevented the RNAi nuclease from degrading
the target mRNA
3. Facts suggest that a nuclease digests dsRNA into small
fragments, which initiate the RNAi process by activating
and guiding the nuclease to the mRNA.
136. Generation of 21-23 nt fragments of RNA in a RNAi-
competent Drosophila embryo extract.
dsRNA of luciferase
reporter genes were added
to the reaction (lanes 2-
10), in the presence or
absence of the
corresponding mRNA. The
dsRNAs were labeled on
the sense, antisense or
both strands. Lanes 11, 12
contained 32
P-labeled,
capped antisense Rluc
RNA.
Fig. 16.38
137. The dsRNA that is added dictates
where the destabilized mRNA is
cleaved.
The dsRNAs A, B, or C were added to
the Drosophila extract together with
a Rr-luc mRNA that is 32
P-labeled at
the 5’ end. The RNA was then
analyzed on a polyacrylamide gel and
autoradiographed.
Results: the products of Rr-luc mRNA
degradation triggered by RNA B are ~100nt
longer than those triggered by RNA C (and
~100 nt longer again for RNA A induced
degradation).
Fig. 16.39
138. High resolution gel analysis of the
products of Rr-luc mRNA
degradation from the previous slide.
Results: the cleavages
occur mainly at 21-23 nt
intervals. There is an
exceptional cleavage only 9
nt from an adjacent cleavage
for RNA C. This cleavage
occurred at a stretch of 7 Us.
14 of 16 cleavage sites were
at a U.
139. Model for RNAi
By “Dicer”
21-23 nt RNAs
Fig. 16.41
ATP-dependent
Helicase?
Active siRNA
complexes.
Very efficient process
because many small
interfering RNAs
(siRNAs) generated from
a larger dsRNA.
140. Biological Significance of RNAi
Most widely held view is that RNAi evolved to
protect the genome from viruses (or other invading
DNAs or RNAs)
Recently, very small (micro) RNAs have been
discovered in several eukaryotes that regulate
developmentally other large RNAs
– May be a new use for the RNAi mechanism besides
defense
142. 1. rRNA, ribosomal RNA
Catalyze protein synthesis by facilitating the binding of tRNA (and
their amino acids) to mRNA.
2. tRNA, transfer RNA
Transport amino acids to mRNA for translation.
3. snRNA, small nuclear RNA
Combine with proteins to form complexes used in RNA processing
(e.g., the splicosome).
143. 1. Synthesis of ribosomal RNA and ribosomes:
1. Cells contain thousands of ribosomes.
2. Consist of two subunits (large and small) in prokaryotes and
eukaryotes, in combination with ribosomal proteins.
3. E. coli 70S model:
50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins
30S subunit = 16S (1,542 nt) + 20 proteins
4. Mammalian 80S model:
60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50
proteins
40S subunit = 18S (1,900 nt) + 35 proteins
5. DNA regions that code for rRNA are called ribosomal DNA (rDNA).
6. Eukaryotes generally have many copies of rRNA genes tandemly
repeated.
144. 1. Synthesis of ribosomal RNA and ribosomes:(continued):
7. Transcription occurs by the same mechanism as protein-coding genes,
but generally using RNA polymerase I.
8. rRNA synthesis requires its own array transcription factors (TFs)
9. Coding sequences for RNA subunits within rDNA genes contain internal
(ITS), external (ETS), and nontranscribed spacers (NTS).
10. ITS units separate the RNA subunits through the pre-rRNA stage,
whereupon ITS & ETS are cleaved out and rRNAs are assembled.
11. Subunits of mature ribosomes are bonded together by H-bonds.
12. Finally, transported to the cytoplasm to initiate protein synthesis.
147. 2. Synthesis of tRNA:
1. tRNA genes also occur in repeated copies throughout the genome,
and may contain introns.
2. Each tRNA (75-90 nt in length) has a different sequence that binds a
different amino acid.
3. Many tRNAs undergo extensive post-transcription modification,
especially those in the mitochondria and chloroplast.
4. tRNAs form clover-leaf structures, with complementary base-pairing
between regions to form four stems and loops.
5. Loop #2 contains the anti-codon, which recognizes
mRNA codons during translation.
6. Same general mechanism using RNA polymerase III, promoters,
unique TFs, plus posttranscriptional modification from pre-tRNA.
148.
149. 3. Synthesis of snRNA (small nuclear RNA):
• Form complexes with proteins used in eukaryotic RNA processing,
e.g., splicing of mRNA after introns are removed.
• Transcribed using RNA polymerase II or III.
150. Reference:
CAIs:
1. Principles of Genetics: Kevin G. McCracken
– http://mercury.bio.uaf.edu/~kevin_mccracken/genetics/
2.Molecular Biology : Profs. Ding Xue and Ravinder
Singh
– http://mcdb.colorado.edu/labs/xue/
3. Molecular Biology : David L. Herrin, Ph.D.
– http://www.esb.utexas.edu/herrin/bio344/
4. Dr. Eric Aamodt
– http://www.sh.lsuhsc.edu/new_curric/mod1_1.html
5.Molecular Biology:5.Molecular Biology: zhenyonglianzhenyonglian
– www.hzau.edu.cn
