Download gene transcription and rna modification

CHAPTER 12
GENE TRANSCRIPTION AND
RNA MODIFICATION
Anatomy of a gene
A typical protein-encoding gene can be divided into the
following elements:
1. Transcriptional control sequences:
bind transcription factors that can activate or inhibit
transcription
2. Promoter: recruits RNA polymerase and marks the start
of the transcribed region
3. Transcript: corresponds to the RNA sequence
Can be further subdivided into:
•
•
•
5' untranslated region (5'UTR): before open reading frame –
often has sequences that control translation
open reading frame (ORF): sequence translated into
polypeptide
3' untranslated region (3'UTR): after ORF; sometime has
sequences that control translation
4. Termination sequences: required to terminate
transcription
Prokaryotic transcriptional regulation
Transcription factor (TF): Any protein that initiates or regulates transcription
In Prokaryotes: promoters are recognised by the basal transcription
apparatus
• This is the complex of TFs that assemble to recruit RNA polymerase
• A protein called σ factor is required for promoter binding
• The exchange of different σ factors and the re-targeting of RNA pol
to a different promoter is the basis of selective gene expression in
prokaryotes
• Basal apparatus + RNA pol holoenzyme = Inititation complex
A. Initiation
RNA polymerase binds to promoter &
opens helix
B. Polymerisation
De novo synthesis using rNTPs as
substrate
Chain elongation in 5’-3’ direction
C. Termination stops at termination
signal
Stages in the process
Initiation:
Closed promoter complex
RNA pol binds the promoter.
Open promoter complex
The DNA is unwound (about 1-1.5 turn of the helix from the -10
region to +2/+3) to allow one strand to act as template.
Initiation
2 NTPs are joined together:
N1TP + N2TP –> pppN1-p-N2 + PPi
• This stage is often thought to be complete after polymerization
of 8-9 nucleotides. This is called promoter clearance: the 
subunit dissociates and RNA pol leaves the promoter.
• Elongation commences
Stages in the process
Elongation:
• The core polymerase continues using the DNA template
strand as a template and adds to the growing RNA
strand.
• Behind the polymerization site is an RNA-DNA duplex
that is only ~1 turn of helix (8-9bp); the other DNA
strand will reform a DNA duplex with the template
strand after that, displacing the RNA.
• A total of ~17 nt of the coding strand is displaced by the
"transcription bubble."
• The transcription bubble will be preceded by positive
supercoils and followed by negative supercoils. Both are
relieved by topoisomerases.
• RNA pol moves at a rate of ~50-90 nt/sec.
Stages in the process
Termination:
– RNA pol is removed from
the gene (otherwise the
entire genome would be
transcribed).
– This usually requires a
secondary structure in the
sequence of the 3'UTR
(most often a stem-loop)
and
• Rho termination factor (dependent)
• a poly-U sequence (independent)
12.3 TRANSCRIPTION IN
EUKARYOTES
• Many of the basic features of gene transcription
are very similar in bacteria and eukaryotes
• However, gene transcription in eukaryotes is
more complex
– Larger organisms
– Cellular complexity
– Multicellularity
Eukaryotic RNA Polymerases

Nuclear DNA is transcribed by three different RNA
polymerases

RNA pol I


Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II

Transcribes all structural genes



Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III


Transcribes all tRNA genes
And the 5S rRNA gene
Genes are first transcribed, and then
translated.
Transcription
 The first step in gene expression is the production of an RNA
copy of the DNA sequence encoding the gene, a process
called transcription.
 To understand the mechanism behind the transcription
process, it is useful to focus first on RNA polymerase, the
remarkable enzyme responsible for carrying it out.
RNA Polymerase
 RNA polymerase is best understood in bacteria.
 Bacterial RNA polymerase is very large and complex,
consisting of five subunits:
1. Two α subunits bind regulatory proteins.
2. β′ subunit binds the DNA template
3. β subunit binds RNA nucleoside subunits.
4. σ subunit recognizes the promoter and initiates synthesis.
 Only one of the two strands of DNA, called the template
strand, is transcribed.
 The strand of DNA that is not transcribed is called the
coding strand.
 The polymerase adds ribonucleotides to the growing 3′ end
of an RNA chain.
 Bacteria contain only one RNA polymerase enzyme, while
eukaryotes have three different RNA polymerases:
1. RNA polymerase I: synthesizes rRNA in the nucleolus.
2. RNA polymerase II: synthesizes mRNA.
3. RNA polymerase III: synthesizes tRNA.
Promoter
 Transcription starts at RNA polymerase binding sites
called promoters on the DNA template strand.
 A promoter is a short sequence that is not itself transcribed
by the polymerase that binds to it.
 Promoters differ widely in efficiency.
 Strong promoters cause frequent initiations of
transcription, as often as every 2 seconds in some bacteria.
 Weak promoters may transcribe only once every 10
minutes.
Initiation
• The binding of RNA polymerase to the
promoter is the first step in gene
transcription.
• In bacteria, a subunit of RNA polymerase
called σ (sigma) recognizes the –10 sequence
in the promoter and binds RNA polymerase
there.
• Importantly, this subunit can detect the –10
sequence without unwinding the DNA double
helix.
• In eukaryotes, the –25 sequence plays a similar
role in initiating transcription, as it is the
binding site for a key protein factor.
• Other eukaryotic factors then bind one after
another, assembling a large and complicated
transcription complex.
• The eukaryotic transcription complex is
described in detail in the following chapter.
Once bound to the promoter, the RNA
polymerase begins to unwind the DNA helix.
Elongation
 Unlike DNA synthesis, a primer is not required.
 The region containing the RNA polymerase, DNA, and
growing RNA transcript is called the transcription bubble
because it contains a locally unwound “bubble” of DNA.
 The transcription bubble moves down the DNA at a constant
rate, about 50 nucleotides per second, leaving the growing
RNA strand protruding from the bubble.
 After the transcription bubble passes, the now transcribed
DNA is rewound as it leaves the bubble.
 Unlike DNA polymerase,
proofreading capability.
RNA
polymerase
has
no
 Transcription thus produces many more copying errors than
replication.
 These mistakes, however, are not transmitted to progeny.
 Most genes are transcribed many times, so a few faulty
copies are not harmful.
Termination
 At the end of a gene are “stop” sequences that cause the
formation of phosphodiester bonds to cease the RNA
polymerase to release the DNA, and the DNA within the
transcription bubble to rewind.
 The simplest stop signal is a series of GC base-pairs followed
by a series of AT base-pairs.
 The RNA transcript of this stop region forms a GC hairpin
followed by four or more U ribonucleotides.
 How does this structure terminate transcription? The hairpin
causes the RNA polymerase to pause immediately after the
polymerase has synthesized it, placing the polymerase
directly over the run of four uracils.
 The pairing of U with DNA’s A is the weakest of the four
hybrid base-pairs and is not strong enough to hold the
hybrid strands together during the long pause.
 Instead, the RNA strand dissociates from the DNA within
the transcription bubble, and transcription stops.
 A variety of protein factors aid hairpin loops in
terminating transcription of particular genes.
12.4 RNA MODIFICATION
• Analysis of bacterial genes in the 1960s and 1970
revealed the following:
– The sequence of DNA in the coding strand corresponds to the
sequence of nucleotides in the mRNA
– This in turn corresponds to the sequence of amino acid in the
polypeptide
• This is termed the colinearity of gene expression
• Analysis of eukaryotic structural genes in the late 1970s
revealed that they are not always colinear with their
functional mRNAs
12.4 RNA MODIFICATION
• Instead, coding sequences, called exons, are interrupted
by intervening sequences or introns
• Transcription produces the entire gene product
– Introns are later removed or excised
– Exons are connected together or spliced
• This phenomenon is termed RNA splicing
– It is a common genetic phenomenon in eukaryotes
– Occurs occasionally in bacteria as well
RNA MODIFICATION
• Aside from splicing, RNA transcripts can be modified in
several ways
– For example
• Trimming of rRNA and tRNA transcripts
• 5’ Capping and 3’ polyA tailing of mRNA transcripts
– See Next Figure….
Trimming

Many nonstructural genes are initially transcribed as a
large RNA

This large RNA transcript is enzymatically cleaved into
smaller functional pieces

Figure 12.14 shows the processing of mammalian
ribosomal RNA
RNA poly I
45s rRNA
Enzymatic cleavage
This processing
occurs in the
nucleolus
Functional RNAs that are
key in ribosome structure
Figure 12.14
Splicing

Three different splicing mechanisms have been
identified




Group I intron splicing
Group II intron splicing
Spliceosome
All three cases involve


Removal of the intron RNA
Linkage of the exon RNA by a phosphodiester bond

Splicing among group I and II introns is termed selfsplicing



Group I and II differ in the way that the intron is
removed and the exons reconnected


Splicing does not require the aid of enzymes
Instead the RNA itself functions as its own ribozyme
Refer to Figure 12.18
Group I and II self-splicing can occur in vitro without the
additional proteins

However, in vivo, proteins known as maturases often enhance
the rate of splicing
Figure 12.18
12-61

In eukaryotes, the transcription
of structural genes, produces a
long transcript known as premRNA

Also as heterogeneous nuclear RNA
(hnRNA)

This RNA is altered by splicing
and other modifications, before it
leaves the nucleus

Splicing in this case requires the
aid of a multicomponent
structure known as the
spliceosome
Figure 12.16
Capping

Most mature mRNAs have a 7-methyl guanosine
covalently attached at their 5’ end


Capping occurs as the pre-mRNA is being synthesized
by RNA pol II


This event is known as capping
Usually when the transcript is only 20 to 25 bases long
As shown in Figure 12.19, capping is a three-step
process
Capping

The 7-methylguanosine cap structure is recognized by
cap-binding proteins

Cap-binding proteins play roles in the



Movement of some RNAs into the cytoplasm
Early stages of translation
Splicing of introns
Tailing

Most mature mRNAs have a string of adenine
nucleotides at their 3’ ends


The polyA tail is not encoded in the gene sequence


This is termed the polyA tail
It is added enzymatically after the gene is completely
transcribed
The attachment of the polyA tail is shown in Figure
12.20
Figure 12.20
Consensus sequence
in higher eukaryotes
From a few dozen adenine
to several hundred
Appears to be important in
the stability of mRNA and
the translation of the
polypeptide
Length varies between
species