Bio 108 Cell Biology lec 6 Regulation of Transcription Initiation
Regulation of Transcription Initiation
-overall process by which the information encoded
in a gene is converted into an observable phenotype
(most commonly production of a protein).
Why- regulate gene expression?
• To adjust to sudden changes
• To conserve energy
• To save resources
Gene control- All of the mechanisms involved in regulating gene
expression. Most common is regulation of transcription, although
mechanisms influencing the processing, stabilization, and translation
of mRNAs help control expression of some genes.
TYPE OF RNA FUNCTION
mRNAs messenger RNAs, code for proteins
rRNAs ribosomal RNAs, form the basic structure of
the ribosome and catalyze protein synthesis
tRNAs transfer RNAs, central to protein synthesis as
adaptors between mRNA and amino acids
snRNAs small nuclear RNAs, function in a variety of
nuclear processes, including the splicing of
snoRNAs small nucleolar RNAs, used to process and
chemically modify rRNAs
Other noncoding RNAs function in diverse cellular processes,
including telomere synthesis, X-chromosome
inactivation, and the transport of proteins into
Table 6-1. Principal Types of RNAs Produced in Cells
Regulation of gene expression allows cells to adapt to changes in their
environments and is responsible for the distinct activities of the multiple
differentiated cell types that make up complex plants and animals.
Control of transcription initiation—the first step—is the most important
mechanism for determining whether or not most genes are expressed and how
much of the encoded mRNAs, and consequently proteins, are produced.
- a process in which a DNA strand provides the information
for the synthesis of an RNA strand.
-enzymes responsible for transcription in both prokaryotic
and eukaryotic cells are called DNA-dependent RNA polymerases
or simply RNA polymerases.
Transcription in Prokaryotes
RNA polymerase- An enzyme that catalyzes the synthesis of RNA.
-the complete enzyme
consists of five subunits: two
α, one β, one β′, and one σ.
-catalyzes the growth of RNA
chains always in the 5′ to 3′
direction (similar to that of DNA
- however, RNA polymerase does
not require a preformed primer
to initiate the synthesis of RNA.
-σ subunit is required to identify
the correct sites for transcription
Promoter -a DNA sequence to which RNA polymerase binds to initiate
Figure 6.2. Sequences of E. coli
promoters E. coli promoters
are characterized by two sets
of sequences located 10 and 35
base pairs upstream of the
transcription start site (+1). The
consensus sequences shown
correspond to the bases most
frequently found in different
*role of σ is to direct the polymerase to promoters by binding specifically
to both the -35 and -10 sequences, leading to the initiation of
transcription at the beginning of a gene
Figure 6.4. Transcription by
E. coli RNA polymerase
a. the polymerase initially
binds nonspecifically to
DNA and migrates along the
molecule until the σ subunit
binds to the -35 and -10
forming a closed-promoter
b. the polymerase then
unwinds (15 bases) DNA
around the initiation site
c. transcription is initiated
by the polymerization of 2
d. σ subunit then
dissociates from the core
polymerase, which migrates
along the DNA and
elongates the growing RNA
RNA Chain Termination - Termination of RNA chain synthesis appears to be
brought about by two types of mechanisms.
In the first type the termination signal appears to be recognized by DNA
itself. RNA polymerase reads an extended poly (A) sequence on DNA. This
results in an RNA transcript with a terminal poly(U) sequence
The second type of termination signal involves an additional protein called
the rho (P) factor. Rho factor is an essential transcription protein in
prokaryotes. In Escherichia coli, it is a ~275 kD hexamer of identical subunits.
Each subunit has an RNA-binding domain and an ATP-hydrolysis domain.
Rho is a member of the family of ATP-dependent hexameric helicases that
function by passing nucleic acids through the hole in the middle of the
hexamer. Rho functions as an ancillary factor for RNA polymerase. Rho-
dependent terminators account for about half of E. coli terminators. The
(rho) factor probably binds to RNA polymerase. It is however, not certain
whether it also, or exclusively, binds to DNA.
1. Intrinsic termination (also called Rho-independent termination) is a
mechanism in both eukaryotes and prokaryotes that causes mRNA transcription
to be stopped. In this mechanism, the mRNA contains a sequence that can base
pair with itself to form a stem-loop structure 7-20 base pairs in length that is also
rich in Cytosine-Guanine base pairs.
Figure 6.5. Transcription
termination The termination
of transcription is signaled by
a GC-rich inverted repeat
followed by four A residues.
The inverted repeat forms a
stable stem-loop structure in
the RNA, causing the RNA to
dissociate from the DNA
2. Rho Dependent Polarity
In Prokaryotes transcription and translation are coupled -- that is, ribosomes
begin translation while the mRNA is being produced.
Coupled transcription and translation. During transcription RNA polymerase (RNAP)
separates the two DNA strands and forms a short region of base pairing between the coding
strand of DNA and the newly synthesized RNA. As the RNA elongates it is displaced as the
single-stranded DNA rehybridizes to form double-stranded DNA. Ribosomes rapidly bind to
the resulting single-stranded RNA and begin protein synthesis.
Rho dependent polarity. The process of Rho
dependent polarity is shown stepwise .Rho
dependent polarity is usually initiated by
premature transcription termination.
When ribosomes encounter a stop (aka
"nonsense") codon, they fall off of the RNA.
Rho factor then interacts with RNAP, causing it to
fall off of the DNA which terminates
transcription. Thus, when translation termination
occurs within a gene it can cause transcriptional
termination, preventing expression of
downstream genes. This process is called
Rho dependent polarity is clearly more
complex than the model shown above.
There is biochemical and genetic evidence
that Rho factor interacts with other
transcription termination factors (for
example, Nus) and the alpha subunit of
Rho factor cannot bind to RNA coated with
ribosomes, but Rho factor binds to specific
regions in naked RNA.
Concept 1: Gene Regulation in Bacteria
Bacteria adapt to changes in their surroundings by using
regulatory proteins to turn groups of genes on and off in
response to various environmental signals.
The DNA of Escherichia coli is sufficient to encode about 4000
proteins, but only a fraction of these are made at any one time. E.
coli regulates the expression of many of its genes according to the
food sources that are available to it.
Concept 2: The Lactose Operon (Jacob-Monod model)
-An operon is a cluster of bacterial genes
along with an adjacent promoter that controls the
transcription of those genes.
When the genes in an operon are transcribed, a single
mRNA is produced for all the genes in that operon. This
mRNA is said to be polycistronic because it carries the
information for more than one type of protein.
• Promoter (DNA) – where RNA polymerase binds
• Operator (DNA) – lies between promoter and
structural genes (regulatory
sequence of DNA that controls
transcription of an operon)
•Repressor (protein) – binds to operator to block
Concept 3: The lac Operator
The operator is a short region of DNA that
lies partially within the promoter and that
interacts with a regulatory protein that controls
the transcription of the operon.
*Here's an analogy. A promoter is like a doorknob, in that the
promoters of many operons are similar. An operator is like the
keyhole in a doorknob,in that each door is locked by only a
specific key, which in this analogy is a specific regulatory protein.
The lac operon of E.coli – an inducible system
includes 3 genes:
1. lacZ –encodes β-galactosidase (catalyzes the hydrolysis
of lactose to glucose and galactose)
2. lacY – encodes lactose permease (carrier protein in the
bacterial plasma membrane that moves the sugar into
3. lacA- encodes thiogalactoside transacetylase (transfers
acetyl group from acetyl-CoA to β-galactosides)
Figure 10-2. Jacob and Monod
model of transcriptional
regulation of the lac operon by
1. When lac repressor binds to
a DNA sequence called the
operator (O), which lies just
upstream of the lacZ gene,
transcription of the operon
by RNA polymerase is
2. 2. Binding of lactose to the
repressor causes a
conformational change in the
repressor, so that it no longer
binds to the operator. RNA
polymerase then is free to
bind to the promoter (P) and
initiate transcription of the
3. the resulting polycistronic
mRNA is translated into the
Concept 4: The lac Regulatory Gene
The regulatory gene lacI produces an mRNA that produces a Lac
repressor protein, which can bind to the operator of the lac operon.
In some texts, the lacI regulatory gene is called the lacI regulator gene. Regulatory genes
are not necessarily close to the operons they affect.
The general term for the product of a regulatory gene is a regulatory protein. The Lac
regulatory protein is called a repressor because it keeps RNA polymerase from
transcribing the structural genes. Thus the Lac repressor inhibits transcription of the lac
Concept 5: The Lac Repressor Protein
In the absence of lactose, the Lac repressor binds
to the operator and keeps RNA polymerase from
transcribing the lac genes.
It would be energetically wasteful for E. coli if the lac genes were
expressed when lactose was not present.
Concept 6: The Effect of Lactose on the lac Operon
When lactose is present, the lac genes are expressed
because allolactose binds to the Lac repressor protein and
keeps it from binding to the lac operator.
Allolactose is an isomer of lactose. Small amounts of allolactose are
formed when lactose enters E. coli.
Allolactose binds to an allosteric site on the repressor protein causing a conformational
change. As a result of this change, the repressor can no longer bind to the operator region
and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.
Concept 7: The lac Inducer: Allolactose
Allolactose is called an inducer because it
turns on, or induces the expression of, the lac
The presence of lactose (and thus allolactose) determines whether or not the Lac
repressor is bound to the operator.
Allolactose binds to an allosteric site on the repressor protein causing a
conformational change. As a result of this change, the repressor can no longer
bind to the operator region and falls off. RNA polymerase can then bind to the
promoter and transcribe the lac genes.
Concept 8: Feedback Control of the lac Operon
When the enzymes encoded by the lac operon
are produced, they break down lactose and
allolactose, eventually releasing the repressor to
stop additional synthesis of lac mRNA.
Messenger RNA breaks down after a relatively short amount of time.
Concept 9: Energy Source Preferences of E. coli
Whenever glucose is present, E. coli
metabolizes it before using alternative energy
sources such as lactose, arabinose, galactose, and
Glucose is the preferred and most frequently available energy source for E. coli. The enzymes
to metabolize glucose are made constantly by E. coli.
When both glucose and lactose are available, the genes for lactose metabolism are
transcribed at low levels.
Only when the supply of glucose has been exhausted does does RNA polymerase start to
transcribe the lac genes efficiently, which allows E. coli to metabolize lactose.
Concept 10: The Effect of Glucose and Lactose on the lac
When both glucose and lactose are present, the genes
for lactose metabolism are transcribed to a small extent.
Maximal transcription of the lac operon occurs only when glucose
is absent and lactose is present. The action of cyclic AMP and a
catabolite activator protein produce this effect.
Positive Control and Catabolite Repression
Concept 11: The Effect of Glucose and Cyclic AMP on the lac
The presence or absence of glucose affects the lac
operon by affecting the concentration of cyclic AMP.
*The concentration of cyclic AMP in E. coli is inversely proportional to
the concentration of glucose: as the concentration of glucose
decreases, the concentration of cyclic AMP increases.
Cyclic AMP is derived from ATP.
Concept 12: The Effect of Lactose in the Absence of Glucose
on the lac Operon
In the presence of lactose and absence of glucose,
cyclic AMP (cAMP) joins with a catabolite activator protein
that binds to the lac promoter and facilitates the
transcription of the lac operon.
In some texts, the catabolite activator protein (CAP) is called the
glucose is low, cAMP
accumulates in the
cell. The binding of
cAMP and the
protein to the lac
binding of RNA
polymerase to the
The lac operon – summary
• In the absence of inducer, the operon is turned off
• Control is exerted by a regulatory protein- the repressor-
that turns the operon off.
• Regulatory genes produce proteins whose sole function is to
regulate the expression of other genes.
• Certain other DNA sequences (operators and promoters) do not
code for proteins, but are binding sites for regulatory or
• Adding inducers turns the operon on.
Operator-repressor control that induces transcription.
The trp operon of E. coli – a repressible system
• In repressible systems, the repressor protein cannot shut off its
operon unless it first binds to a corepressor, which may be
either the metabolic end product itself (tryptophan in this
case) or an analog of it.
Operator-repressor control that represses transcription
1. Regulatory gene r produces an inactive
repressor, which cannot bind to the
PTrp o e d c b a
e d c b a
Enzymes of the tryptophan
2. RNA polymerase transcribes the
structural genes. Translation
makes the enzymes of the
tryptophan synthesis pathway.
PTrp o e d c b a
1. Tryptophan binds the
2. … which then binds
to the operator.
3. Trp blocks RNA pol from
binding and transcribing the
structural genes, preventing
synthesis of trp pathway
Inducible vs. Repressible systems
• in inducible systems, the substrate of a metabolic
pathway (the inducer) interacts with a regulatory protein
(the repressor) to render it incapable of binding to the
operator, thus allowing transcription
• in repressible systems, the product of a metabolic
pathway (the corepressor) interacts with the regulatory
protein to make it capable of binding to the operator,
thus blocking the transcription
• In both kinds of systems, the regulatory molecule
functions by binding to the operator.
Examples of Negative control of transcription:
a. inducible lac system
b. repressible trp system
•The two operator-repressor systems because the
regulatory molecule (the repressor) in each case prevents
Example of Positive control of transcription:
a. promoter-catabolite repression system
• because the regulatory molecule (the CRP-cAMP complex)
Transcription in Eukaryotes
Transcription Initiation in Eucaryotes Requires Many Proteins
- eucaryotic nuclei have three RNA pol (bacteria only one):
a. RNA polymerase I
b. RNA polymerase II
c. and RNA polymerase III
* structurally similar to one another but they transcribe different types
TYPE OF POLYMERASE GENES TRANSCRIBED
RNA polymerase I 5.8S, 18S, and 28S rRNA genes
RNA polymerase II all protein-coding genes, plus
snoRNAgenes and some snRNA genes
RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA
genes and genes for other small RNAs
Table 6-2. The Three RNA Polymerases in Eucaryotic Cells
Transcription factor (TF) - General term for
any protein, other than RNA polymerase,
required to initiate or regulate transcription in
eukaryotic cells. General factors, required for
transcription of all genes, participate in
formation of the transcription-initiation
complex near the start site. Specific factors
stimulate (or repress) transcription of
particular genes by binding to their regulatory
Several important differences in the way in which the bacterial and
eucaryotic enzymes function:
1. While bacterial RNA polymerase (with σ factor as one of its subunits) is able
to initiate transcription on a DNA template in vitro without the help of
additional proteins, eucaryotic RNA polymerases cannot. They require the
help of a large set of proteins called general transcription factors, which must
assemble at the promoter with the polymerase before the polymerase can
2. Eucaryotic transcription initiation must deal with the packing of DNA into
nucleosomes and higher order forms of chromatin structure, features absent
from bacterial chromosomes.
RNA Polymerase II Requires General Transcription Factors
General transcription factor -Any of the proteins whose assembly around the
TATA box is required for the initiation of transcription of most eucaryotic
TATA box -Consensus sequence in the promoter region of many eucaryotic
genes that binds a general transcription factor and hence specifies the
position at which transcription is initiated.
TATA-binding protein (TBP) -A basal transcription factor that binds directly to the TATA
TFIID (Transcription factorIID) is itself composed of multiple subunits, including
the TATA-binding protein (TBP), which binds specifically to the TATAA consensus
sequence, and 10-12 other polypeptides, called TBP-associated factors (TAFs)
Figure 6.14. RNA polymerase
II holoenzyme The
holoenzyme consists of a
preformed complex of RNA
polymerase II, the general
transcription factors TFIIB,
TFIIE, TFIIF, and TFIIH, and
several other proteins that
activate transcription. This
complex can be recruited
directly to a promoter via
interaction with TFIID (TBP +
Figure 6-16. Initiation of transcription of a
eucaryotic gene by RNA polymerase II. To
begin transcription, RNA polymerase
requires a number of general transcription
factors (called TFIIA, TFIIB, and so on). (A)
The promoter contains a DNA sequence
called the TATA box, which is located 25
nucleotides away from the site at which
transcription is initiated. (B) The TATA box is
recognized and bound by transcription
factor TFIID, which then enables the
adjacent binding of TFIIB (C). For simplicity
the DNA distortion produced by the binding
of TFIID (see Figure 6-18) is not shown. (D)
The rest of the general transcription factors,
as well as the RNA polymerase itself,
assemble at the promoter. (E) TFIIH then
uses ATP to pry apart the DNA double helix
at the transcription start point, allowing
transcription to begin. TFIIH also
phosphorylates RNA polymerase II,
changing its conformation so that the
polymerase is released from the general
factors and can begin the elongation phase
TFIIH is a multisubunit factor that appears to play at least two
a. First, two subunits of TFIIH are helicases, which may
unwind DNA around the initiation site. (These subunits of TFIIH are
also required for nucleotide excision repair).
b. Another subunit of TFIIH is a protein kinase that
phosphorylates repeated sequences present in the C-terminal
domain of the largest subunit of RNA polymerase II.
Phosphorylation of these sequences is thought to release the
polymerase from its association with the initiation complex,
allowing it to proceed along the template as it elongates the
growing RNA chain
Figure 6.12. Formation of a
polymerase II transcription
complex Many polymerase II
promoters have a TATA box
(consensus sequence TATAA) 25
to 30 nucleotides upstream of
the transcription start site. This
sequence is recognized by
transcription factor TFIID, which
consists of the TATA-binding
protein (TBP) and TBP-
associated factors (TAFs).
TFIIB(B) then binds to TBP,
followed by binding of the
polymerase in association with
TFIIF(F). Finally, TFIIE(E) and
TFIIH(H) associate with the
Figure 10-6. DNase I footprinting, a
common technique for identifying
protein-binding sites in DNA.
a. A DNA fragment is labeled at one
end with 32P (red dot) as in the
Maxam-Gilbert sequencing method.
b. Portions of the sample then are
digested with DNase I in the
presence and absence of a protein
that binds to a specific sequence in
(Bottom) Diagram of hypothetical
autoradiogram of the gel for the minus
protein sample above reveals bands
corresponding to all possible
fragments produced by DNase I
cleavage (−lane). In the sample
digested in the presence of a DNA-
binding protein, two bands are missing
(+lane); these correspond to the DNA
region protected from digestion by
bound protein and are referred to as
the footprint of that protein.
Polymerase II Also Requires Activator, Mediator, and Chromatin-
DNA in eucaryotic cells is packaged into nucleosomes, which are further arranged
in higher-order chromatin structures. As a result, transcription initiation in a
eucaryotic cell is more complex and requires more proteins
1. gene regulatory proteins known as transcriptional activators bind to
specific sequences in DNA and help to attract RNA polymerase II to the start point
of transcription (This attraction is needed to help the RNA polymerase and the
general transcription factors in overcoming the difficulty of binding to DNA that is
packaged in chromatin).
2. eucaryotic transcription initiation in vivo requires the presence of a
protein complex known as the mediator, which allows the activator proteins to
communicate properly with the polymerase II and with the general transcription
3. transcription initiation in the cell often requires the local recruitment of
chromatin-modifying enzymes, including chromatin remodeling complexes and
histone acetylases (both types of enzymes can allow greater accessibility to the
DNA present in chromatin, and so, they facilitate the assembly of the transcription
initiation machinery onto DNA).
A typical eucaryotic gene
has many activator
proteins, which together
determine its rate and
pattern of transcription.
Sometimes acting from a
distance of several
thousand nucleotide pairs
(indicated by the dashed
DNA molecule), these gene
regulatory proteins help
RNA polymerase, the
general factors, and the
mediator all to assemble at
*activators attract ATP-
remodeling complexes and
Protein-coding genes have
•exons whose sequence encodes the polypeptide;
•introns that will be removed from the mRNA before it is
•a transcription start site
the basal or core promoter located within about 40 bp of
the start site
an "upstream" promoter, which may extend over as many
as 200 bp farther upstream
Adjacent genes (RNA-coding as well as protein-coding) are
often separated by an insulator which helps them avoid cross-
talk between each other's promoters and enhancers (and/or
Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that
are thousands of base pairs away from the gene they control. Binding increases the
rate of transcription of the gene.
Enhancers can be located upstream, downstream, or even within the gene they
How does the binding of a protein to an enhancer regulate the transcription of a
gene thousands of base pairs away?
One possibility is that enhancer-binding proteins — in addition to their DNA-
binding site, have sites that bind to transcription factors ("TF") assembled at the
promoter of the gene.
This would draw the DNA into a loop (as shown in the figure).
Michael R. Botchan (who kindly supplied these
electron micrographs) and his colleagues have
produced visual evidence of this model of
enhancer action. They created an artificial DNA
•several (4) promoter sites for Sp1 about 300
bases from one end. Sp1 is a zinc-finger
transcription factor that binds to the sequence 5'
GGGCGG 3' found in the promoters of many genes,
especially "housekeeping" genes.
•several (5) enhancer sites about 800 bases from
the other end. These are bound by an enhancer-
binding protein designated E2.
•1860 base pairs of DNA between the two.
When these DNA molecules were added to a
mixture of Sp1 and E2, the electron microscope
showed that the DNA was drawn into loops with
"tails" of approximately 300 and 800 base pairs.
At the neck of each loop were two distinguishable
globs of material, one representing Sp1 (red), the
other E2 (blue) molecules. (The two micrographs
are identical; the lower one has been labeled to
show the interpretation.)
Artificial DNA molecules lacking
either the promoter sites or the
enhancer sites, or with mutated
versions of them, failed to form
loops when mixed with the two
Silencers are control regions of DNA that, like enhancers, may be located
thousands of base pairs away from the gene they control. However, when
transcription factors bind to them, expression of the gene they control is
As you can see above, enhancers can turn on promoters of genes located
thousands of base pairs away. What is to prevent an enhancer from
inappropriately binding to and activating the promoter of some other gene in
the same region of the chromosome?
One answer: an insulator.
•stretches of DNA (as few as 42 base pairs may do the trick)
•located between the
enhancer(s) and promoter or
silencer(s) and promoter
of adjacent genes or clusters of adjacent genes.
Their function is to prevent a gene from being influenced by the activation (or
repression) of its neighbors.
The enhancer for the promoter of the gene for the delta chain of the gamma/delta
T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain
of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose
between one or the other. There is an insulator between the alpha gene promoter
and the delta gene promoter that ensures that activation of one does not spread
over to the other.
All insulators discovered so far in vertebrates work only when bound by a protein
designated CTCF ("CCCTC binding factor"; named for a nucleotide sequence
found in all insulators). CTCF has 11 zinc fingers.
Another example: In mammals (mice, humans, pigs), only the allele for insulin-
like growth factor-2 (IGF2) inherited from one's father is active; that inherited
from the mother is not — a phenomenon called imprinting.
The mechanism: the mother's allele has an insulator between the IGF2 promoter
and enhancer. So does the father's allele, but in his case, the insulator has been
methylated. CTCF can no longer bind to the insulator, and so the enhancer is
now free to turn on the father's IGF2 promoter.
Many of the commercially-important varieties of pigs have been bred to
contain a gene that increases the ratio of skeletal muscle to fat. This gene has
been sequenced and turns out to be an allele of IGF2, which contains a single
point mutation in one of its introns. Pigs with this mutation produce higher
levels of IGF2 mRNA in their skeletal muscles (but not in their liver).
This tells us that:
•Mutations need not be in the protein-coding portion of a gene in order to
affect the phenotype.
•Mutations in non-coding portions of a gene can affect how that gene is
regulated (here, a change in muscle but not in liver).
Transcription by RNA Polymerases I and III
All three RNA polymerases, however, require additional transcription
factors to associate with appropriate promoter sequences. Furthermore,
although the three different polymerases in eukaryotic cells recognize
distinct types of promoters, a common transcription factor—the TATA-
binding protein (TBP)—appears to be required for initiation of transcription
by all three enzymes.
RNA polymerase I is devoted solely to the transcription of ribosomal RNA
genes, which are present in tandem repeats.
Figure 6.15. The
ribosomal RNA gene The
ribosomal DNA (rDNA) is
transcribed to yield a large
RNA molecule (45S pre-
rRNA), which is then
cleaved into 28S, 18S, and
Figure 6.16. Initiation
of rDNA transcription
factors, UBF and SL1,
bind cooperatively to
the rDNA promoter
and recruit RNA
polymerase I to form
an initiation complex.
One subunit of SL1 is
UBF (upstream binding
SL1 (selectivity factor 1)
-promoter for ribosomal RNA genes does not contain a TATA box,
TBP does not bind to specific promoter sequences. Instead, the
association of TBP with ribosomal RNA genes is mediated by the
binding of other proteins in the SL1 complex to the promoter, a
situation similar to the association of TBP with the Inr sequences
of polymerase II genes that lack TATA boxes.
-genes for tRNAs, 5S rRNA, and some of the small RNAs
involved in splicing and protein transport are transcribed
by polymerase III.
-these genes are characterized by promoters that lie
within, rather than upstream of, the transcribed
Figure 6.17. Transcription of
polymerase III genes The
promoters of 5S rRNA and tRNA
genes are downstream of the
transcrip-tion initiation site.
Transcription of the 5S rRNA
gene is initiated by the binding
of TFIIIA, followed by the binding
of TFIIIC, TFIIIB, and the
polymerase. The tRNA
promoters do not contain a
binding site for TFIIIA, and TFIIIA
is not required for their
transcription. Instead, TFIIIC
initiates the transcription of
tRNA genes by binding to
promoter sequences, followed
by the association of TFIIIB and
polymerase. The TATA-binding
protein (TBP) is a subunit of
RNA Processing and Turnover
Most newly synthesized RNAs must be modified in various ways
to be converted to their functional forms except bacterial
-the primary transcripts of both rRNAs and tRNAs must undergo
a series of processing steps in prokaryotic as well as eukaryotic
Processing of Ribosomal and Transfer RNAs
basic processing of rRNA and tRNAs in prokaryotic and eukaryotic
cells is similar, as might be expected given the fundamental roles
of these RNAs in protein synthesis.
*eukaryotes have four species of ribosomal RNAs, three of which (the 28S, 18S,
and 5.8S rRNAs) are derived by cleavage of a single long precursor transcript,
called a pre-rRNA
*prokaryotes have three ribosomal RNAs (23S, 16S, and 5S), which are
equivalent to the 28S, 18S, and 5S rRNAs of eukaryotic cells and are also formed
by the processing of a single pre-rRNA transcript.
pre-rRNA -The primary transcript, which is cleaved to form individual ribosomal
RNAs (the 28S, 18S, and 5.8S rRNAs of eukaryotic cells).
Figure 6.37. Processing of
ribosomal RNAs Prokaryotic
cells contain three rRNAs (16S,
23S, and 5S), which are formed
by cleavage of a pre-rRNA
transcript. Eukaryotic cells
(e.g., human cells) contain four
rRNAs. One of these (5S rRNA)
is transcribed from a separate
gene; the other three (18S,
28S, and 5.8S) are derived from
a common pre-rRNA. Following
cleavage, the 5.8S rRNA (which
is unique to eukaryotes)
becomes hydrogen-bonded to
Figure 6.38. Processing of transfer RNAs
(A) Transfer RNAs are derived from pre-
tRNAs, some of which contain several
individual tRNA molecules. Cleavage at
the 5′ end of the tRNA is catalyzed by the
RNase P ribozyme; cleavage at the 3′ end
is catalyzed by a conventional protein
RNase. A CCA terminus is then added to
the 3′ end of many tRNAs in a
posttranscriptional processing step.
Finally, some bases are modified at
characteristic positions in the tRNA
molecule. In this example, these
modified nucleosides include
dihydrouridine (DHU), methylguanosine
(mG), inosine (I), ribothymidine (T), and
pseudouridine (y). (B) Structure of
modified bases. Ribothymidine,
dihydrouridine, and pseudouridine are
formed by modification of uridines in
tRNA. Inosine and methylguanosine are
formed by the modification of
tRNAs in both bacteria and
eukaryotes are synthesized
as longer precursor
some of which contain
several individual tRNA
pre-tRNA The primary
transcript, which is cleaved
to form transfer RNAs.
In bacteria, some tRNAs are
included in the pre-rRNA
RNase P - A ribozyme that cleaves the 5´ end of pre-tRNAs.
- consists of RNA and protein molecules, both of which are
required for maximal activity.
ribozyme An RNA enzyme.
Note: an unusual aspect of tRNA processing is the extensive
modification of bases in tRNA molecules. Approximately 10% of the
bases in tRNAs are altered to yield a variety of modified nucleotides
at specific positions in tRNA molecules (see Figure 6.38). The
functions of most of these modified bases are unknown, but some
play important roles in protein synthesis by altering the base-pairing
properties of the tRNA molecule
Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing
Figure 6-21. Summary of the
steps leading from gene to
protein in eucaryotes and
bacteria. The final level of a
protein in the cell depends on the
efficiency of each step and on the
rates of degradation of the RNA
and protein molecules. (A) In
eucaryotic cells the RNA molecule
produced by transcription alone
(sometimes referred to as the
primary transcript) would contain
both coding (exon) and
noncoding (intron) sequences.
Before it can be translated into
protein, the two ends of the RNA
are modified, the introns are
removed by an enzymatically
catalyzed RNA splicing reaction,
and the resulting mRNA is
transported from the nucleus to
Figure 6-23. The “RNA factory” concept
for eucaryotic RNA polymerase II. Not
only does the polymerase transcribe DNA
into RNA, but it also carries pre-mRNA-
processing proteins on its tail, which are
then transferred to the nascent RNA at
the appropriate time. There are many
RNA-processing enzymes, and not all
travel with the polymerase. For RNA
splicing, for example, only a few critical
components are carried on the tail; once
transferred to an RNA molecule, they
serve as a nucleation site for the
remaining components. The RNA-
processing proteins first bind to the RNA
polymerase tail when it is phosphorylated
late in the process of transcription
initiation). Once RNA polymerase II
finishes transcribing, it is released from
DNA, the phosphates on its tail are
removed by soluble phosphatases, and it
can reinitiate transcription. Only this
dephosphorylated form of RNA
polymerase II is competent to start RNA
synthesis at a promoter.