LECTURE 3
Gene Transcription
and RNA
Modification
(Chapter 12)
1
INTRODUCTION
• The term gene has many definitions
• For this class, a gene is a segment of DNA
used to make a product that plays a
functional role in the cell
– either an RNA or a polypeptide
• Transcription is the first step in gene
expression
2
• Transcription: (Verb) The act or
process of making a copy
– Example: Court reporter hears the
witness speaking in English and types a
written copy, in English, of the witness’
statements.
• Translation: Express the meaning of
words or text in another language
• Dogma: A principle or set of
principles laid down by an authority
as incontrovertibly true
Court reporter
transcribing court
testimony
3
TRANSCRIPTION
• In genetics, the term refers to the copying of a
DNA sequence into an RNA sequence
– Only one strand is copied
• The structure of DNA is not altered as a result
of this process
– It continues to store information and can be
transcribed again and again and again
4
1. Check out
2. Make many
copies of the
same page
3. Return unaltered
4. Distribute and
incite a riot!
5
Gene Expression

Structural genes encode the amino acid
sequence of a polypeptide




Transcription of a structural gene produces
messenger RNA, usually called mRNA
The mRNA nucleotide sequence determines the
amino acid sequence of a polypeptide during
translation
The synthesis of functional proteins determines an
organisms traits
This path from gene to trait is called the central
dogma of genetics

Refer to Figure 12.1
6
The central dogma of genetics
DNA replication: makes DNA copies that are transmitted
from cell to cell and from parent to
offspring.
Gene
Chromosomal DNA: stores information in
units called genes.
Transcription: produces an RNA copy of a gene.
Messenger RNA: a temporary copy of a gene
that contains information to
make a polypeptide.
Translation: produces a polypeptide using the
information in mRNA.
Polypeptide: becomes part of a functional protein
that contributes to an organism's traits.
Figure 12.1
7
Is this simplistic?
8
12.1 OVERVIEW OF
TRANSCRIPTION
• Gene expression is the overall process by
which the information within a gene is used to
produce a functional product which can, in
concert with environmental factors, determine a
trait
– Or: How does a book result in a riot?
9
The Stages of Transcription

Transcription occurs in three stages




Initiation
Elongation
Termination
These steps involve protein-DNA interactions

Proteins such as RNA polymerase interact with DNA
sequences
10
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DNA of a gene
Transcription
Promoter
Terminator
Initiation: The promoter functions as a recognition
site for transcription factors (not shown). The transcription
factor(s) enables RNA polymerase to bind to the promoter.
Following binding, the DNA is denatured into a bubble
known as the open complex.
5′ end of growing
RNA transcript
Open complex
RNA polymerase
Elongation/synthesis of the RNA transcript:
RNA polymerase slides along the DNA
in an open complex to synthesize RNA.
Termination: A terminator is reached that causes RNA
polymerase and the RNA transcript to dissociate from
the DNA.
Completed RNA
transcript
Figure 12.3
RNA
polymerase
11
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12
RNA Transcripts Have
Different Functions

Once they are made, RNA transcripts play different
functional roles


Well over 90% of all genes are structural genes
which are transcribed into mRNA


Refer to Table 12.1
Final functional products are polypeptides
The other RNA molecules in Table 12.1 are never
translated

Final functional products are RNA molecules
13
RNA Transcripts Have Different
Functions

The RNA transcripts from nonstructural genes are
not translated



They do have various important cellular functions
They can still confer traits
In some cases, the RNA transcript becomes part of a
complex that contains protein subunits

For example
 Ribosomes
 Spliceosomes
 Signal recognition particles
14
You don’t need to
memorize this slide –
however, note how
many different types of
functional RNA
molecules exist and
how many different
types of functions they
perform!
15
12.2 TRANSCRIPTION IN
BACTERIA
• Our molecular understanding of gene transcription
came from studies involving bacteria and
bacteriophages
• Indeed, much of our knowledge comes from
studies of a single bacterium
– E. coli, of course
• In this section we will examine the three steps of
transcription as they occur in bacteria
16
Promoters

Promoters are DNA sequences that “promote” gene
expression


More precisely, they direct the exact location for the
initiation of transcription
Promoters are typically located just upstream of the
site where transcription of a gene actually begins

The bases in a promoter sequence are numbered in
relation to the transcription start site

Refer to Figure 12.4
17
Most of the promoter region is
labeled with negative numbers
Bases preceding the
start site are
numbered in a
negative direction
There is no base
numbered 0
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Coding strand
Transcriptional
start site
Promoter region
–35 sequence
16 –18 bp
–10 sequence
+1
5′
T TGA CA
AACTGT
TA TAA T
ATATTA
3′
A
T
3′
5′
Template strand
Bases to the right are
numbered in a
positive direction
5′
3′
A
RNA
Transcription
Figure 12.4 The conventional numbering system of promoters
18
The promoter may span a large
region, but specific short
sequence elements are
particularly critical for promoter
recognition and activity level
Sequence elements that play
a key role in transcription
Coding strand
Transcriptional
start site
Promoter region
–35 sequence
16 –18 bp
–10 sequence
+1
5′
T TGA CA
AACTGT
TA TAA T
ATATTA
3′
A
T
3′
Template strand
5′
Sometimes termed the
Pribnow box, after its
discoverer
5′
3′
A
RNA
Transcription
Figure 12.4 The conventional numbering system of promoters
19
Initiation of Bacterial Transcription

RNA polymerase is the enzyme that catalyzes the
synthesis of RNA

In E. coli, the RNA polymerase holoenzyme is
composed of

Core enzyme


Sigma factor


Five subunits = a2bb’
One subunit = s
These subunits play distinct functional roles
20
Initiation of Bacterial Transcription

The RNA polymerase holoenzyme binds loosely to
the DNA

It then scans along the DNA, until it encounters a
promoter region

When it does, the sigma factor recognizes both the –35
and –10 regions


A region within the sigma factor that contains a helix-turn-helix
structure is involved in a tighter binding to the DNA
Refer to Figure 12.6
21
Binding of sfactor protein to DNA double helix
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α helices
binding to the
major groove
Amino acids within the
a helices hydrogen
bond with bases in the
-35 and -10 promoter
sequences
Figure 12.6
Turn
22

The binding of the RNA polymerase to the promoter
forms the closed complex

Then, the open complex is formed when the
TATAAT box in the -10 region is unwound

A short RNA strand is made within the open
complex

The sigma factor is released at this point


This marks the end of initiation
The core enzyme now slides down the DNA to
synthesize an RNA strand

This is known as the elongation phase
23
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RNA polymerase
Promotor region
σ factor
–35
–10
After sliding along the DNA, σ
factor recognizes a promoter, and
RNA polymerase holoenzyme
forms a closed complex.
–35
RNA polymerase
holoenzyme
–10
Closed complex
An open complex is formed, and
a short RNA is made.
–35
–10
Open complex
σ factor is released, and the
core enzyme is able to proceed
down the DNA.
RNA polymerase
–35
core enzyme
–10
σ factor
Figure 12.7
RNA transcript
24
Character
Played By
A shy female college student
A cute dude
A helpful friend
Dr. Ballard
25
Elongation in Bacterial Transcription

The RNA transcript is synthesized during the
elongation stage

The DNA strand used as a template for RNA
synthesis is termed the template strand

The opposite DNA strand is called the coding strand

It has the same base sequence as the RNA transcript

Except that T in DNA corresponds to U in RNA
26


In transcription, RNA polymerase reads only one
strand of the DNA
 It reads the template strand
 It moves along the template strand 3’ to 5’
The RNA polymerase simultaneously makes a RNA
copy of the template strand’s complementary
partner
 The partner strand is called the coding strand
 The new mRNA molecule is made in the 5’ to 3’
direction
 The orientation and sequence of the mRNA is
identical to the coding strand (except U’s for T’s)
27
28
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29
Elongation in Bacterial Transcription

The open complex formed by the action of RNA
polymerase is about 17 bases long

Behind the open complex, the DNA rewinds back into a
double helix

On average, the rate of RNA synthesis is about 43
nucleotides per second!

Figure 12.8 depicts the key points in the synthesis of
an RNA transcript
30
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Coding
strand
T
AU
G C
T A
5′
Coding
strand
Template
strand
3′
C
A
G
CG
Template strand
3′
Rewinding of DNA
RNA polymerase
Open complex
Unwinding of DNA
RNA
Direction of
transcription
5′
3′
RNA–DNA
hybrid
region
Similar to the
synthesis of DNA
via DNA polymerase
Key points:
5′
Nucleotide being
added to the 3′
end of the RNA
Nucleoside
triphosphates
• RNA polymerase slides along the DNA, creating an open
complex as it moves.
• The DNA strand known as the template strand is used to make a
complementary copy of RNA as an RNA–DNA hybrid.
• RNA polymerase moves along the template strand in a 3′ to 5′ direction,
and RNA is synthesized in a 5′ to 3′ direction using nucleoside
triphosphates as precursors. Pyrophosphate is released (not shown).
Figure 12.8
• The complementarity rule is the same as the AT/GC rule except
that U is substituted for T in the RNA.
31
Termination of Bacterial
Transcription

Termination is the end of RNA synthesis

It occurs when the short RNA-DNA hybrid of the open
complex is forced to separate


This releases the newly made RNA as well as the RNA polymerase
E. coli has two different mechanisms for termination

1. rho-dependent termination


Requires a protein known as r (rho)
2. rho-independent termination

Does not require r
32
ρ recognition site (rut)
Terminator
5′
rut
rho utilization
site
3′
ρ recognition
site in RNA
Rho protein is
a helicase
ρ protein binds to the
rut site in RNA and moves
toward the 3′ end.
5′
3′
ρ protein
RNA polymerase reaches the
terminator. A stem-loop
causes RNA polymerase
to pause.
5′
Terminator
3′
Stem-loop
RNA polymerase pauses
due to its interaction with
the stem-loop structure. ρ
protein catches up to the open
complex and separates the
RNA-DNA hybrid.
3′
5′
Figure 12.10 r-dependent termination
33
•
r-independent termination is facilitated by two sequences in the RNA
– 1. A uracil-rich sequence located at the 3’ end of the RNA
– 2. A stem-loop structure upstream of the uracil-rich sequence
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U-rich RNA in
the RNA-DNA
hybrid
5′
Stem-loop that causes
RNA polymerase to pause
NusA
Stabilizes
the RNA pol
pausing
While RNA polymerase pauses,
the U-rich sequence is not able to
hold the RNA-DNA hybrid together.
Termination occurs.
Terminator
URNA-ADNA hydrogen
bonds are relatively
weak
No protein is required to physically
remove the RNA from the DNA
This type of termination
is also called intrinsic
5′
Figure 12.11 r-independent termination
U
U
U
U 3′
34
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, more complex cells (organelles)
– Added cellular complexity means more genes that
encode proteins are required
– Multicellularity adds another level of regulation
• express genes only in the correct cells at the proper time
35
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 gene
And the 5S rRNA gene
36
Sequences of Eukaryotic
Structural Genes

Eukaryotic promoter sequences are more variable
and often more complex than those of bacteria

For structural genes, at least three features are
found in most promoters

Regulatory elements
TATA box
Transcriptional start site

Refer to Figure 12.13


37
Figure 12.13
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Core promoter
Coding-strand sequences:
–100
Common location for
regulatory elements such
as GC and CAAT boxes
–50
TATA box
Transcriptional
start site
TATAAA
Py2CAPy5
–25
DNA
Usually an
adenine
+1
Transcription
• The core promoter is relatively short
– It consists of the TATA box and transcriptional start site
• Important in determining the precise start point for transcription
• The core promoter by itself produces a low level of
transcription
– This is termed basal transcription
38
Figure 12.13
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Core promoter
Coding-strand sequences:
–100
Common location for
regulatory elements such
as GC and CAAT boxes
–50
DNA
TATA box
Transcriptional
start site
TATAAA
Py2CAPy5
–25
+1
Transcription
• Regulatory elements are short DNA sequences that affect the
binding of RNA polymerase to the promoter
• Transcription factors (proteins) bind to these elements and
influence the rate of transcription
– There are two types of regulatory elements
• Enhancers
– Stimulate transcription
• Silencers
– Inhibit transcription
– They vary widely in their locations but are often found in the
–50 to –100 region
39
Sequences of Eukaryotic Structural
Genes

Factors that control gene expression can be divided
into two types, based on their “location”

cis-acting elements



DNA sequences that exert their effect only over a
particular gene
Example: TATA box, enhancers and silencers
trans-acting elements

Regulatory proteins that bind to such DNA sequences
40
RNA Polymerase II and its
Transcription Factors

Three categories of proteins are required for basal
transcription to occur at the promoter




RNA polymerase II
Five different proteins called general transcription factors
(GTFs)
A protein complex called mediator (we won’t go over this)
Figure 12.14 shows the assembly of transcription
factors and RNA polymerase II at the TATA box
41
Figure 12.14
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TFIID binds to the TATA box. TFIID is
a complex of proteins that includes the
TATA-binding protein (TBP) and several
TBP-associated factors (TAFs).
TFIID
TATA box
You don’t need to
memorize the binding
order but you should
know that several
different general
transcription factors
must bind in order to
recruit RNA
polymerase to the
promoter and start its
action
TFIIB binds to TFIID.
TFIID
TFIIB
TFIIB acts as a bridge to bind
RNA polymerase II and TFIIF.
TFIID
TFIIF
RNA polymerase II
TFIIE and TFIIH bind to RNA
polymerase II to form a preinitiation
or closed complex.
Preinitiation complex
TFIID
TFIIF
TFIIH acts as a helicase to form an
open complex. TFIIH also phosphorylates
the CTD domain of RNA polymerase II.
CTD phosphorylation breaks the contact
between TFIIB and RNA polymerase II.
TFIIB, TFIIE, and TFIIH are released.
Released after the
open complex is
formed
A closed complex
TFIID
TFIIF
Open complex
TFIIB
TFIIE
RNA pol II can now
proceed to the
elongation stage
PO4
PO4
CTD domain of
RNA polymerase II
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42
RNA Pol II transcriptional termination
• Pre-mRNAs are modified by cleavage near
their 3’ end with subsequent attachment of a
string of adenines
• Transcription terminates 500 to 2000
nucleotides downstream from the poly A
signal
• There are two models for termination
– Further research is needed to determine if either,
or both are correct (we won’t cover this)
43
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
– The sequence of codons in the mRNA provides the
instructions for the sequence of amino acids 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
44
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
45
12.4 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
– Refer to Table 12.3
46
Focus
your
attention
here
47
Splicing

Three different splicing mechanisms have been
identified




Group I intron splicing
Group II intron splicing
Spliceosome (we’ll focus on this mechanism)
All three cases involve


Removal of the intron RNA
Linkage of the exon RNA by a phosphodiester bond
48
49



In eukaryotes, the transcription
of structural genes produces a
long transcript known as
pre-mRNA
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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
Intron removed via spliceosome
(very common in eukaryotes)
Intron
Spliceosome
P
O
A
O
5′
Exon 1
CH2
H
2′
H
H
O
O
H
P
Exon 2
3′
P
O
A
O
CH2
H
H 2
H
O
O
P
5′
3′
P
3′
OH
P
O
A
O
H 2′
O
CH2
H
H
O
P
5′
P
3′
mRNA
(c) Pre-mRNA
Figure 12.20
50
Pre-mRNA Splicing

The spliceosome is a large complex that splices
pre-mRNA

It is composed of several subunits known as
snRNPs (pronounced “snurps”)

Each snRNP contains small nuclear RNA and a set of
proteins
51
Pre-mRNA Splicing

The subunits of a spliceosome carry out several
functions

1. Bind to an intron sequence and precisely recognize
the intron-exon boundaries

2. Hold the pre-mRNA in the correct configuration

3. Catalyze the chemical reactions that remove introns
and covalently link exons
52

Intron RNA is defined by particular sequences within the
intron and at the intron-exon boundaries

The consensus sequences for the splicing of mammalian
pre-mRNA are shown in Figure 12.21
Corresponds to the boxed
adenine in Figure 12.22
Sequences shown in bold
are highly conserved
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Exon
5′
Intron
A/
CGGU
Pu AGUA
5′ splice site
Figure 12.21

Exon
UACUUAUCC
Py12N Py AGG
Branch site
3′ splice site
3′
Serve as recognition sites for the
binding of the spliceosome
The pre-mRNA splicing mechanism is shown in Figure 12.22
53
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Exon 1
Exon 2
A
GU
5′
AG
Branch site
5′ splice site
3′
3′ splice site
U1 binds to 5′ splice site.
U2 binds to branch site.
U1 snRNP
U2 snRNP
A
5′
3′
U4/U6 and U5 trimer binds. Intron
loops out and exons are brought
closer together.
Intron loops out and
exons brought closer
together
A
U2
U4/U6 snRNP
U1
5′
Figure 12.22
U5 snRNP
3′
54
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5′ splice site is cut.
5′ end of intron is connected to the
A in the branch site to form a lariat.
U1 and U4 are released.
Cleavage may be
catalyzed by snRNA
molecules within U2
and U6
U1
U4
U2
A
U6
U5
5′
3′
3′ splice site is cut.
Exon 1 is connected to exon 2.
The intron (in the form of a lariat)
is released along with U2, U5,
and U6. The intron will be degraded.
Intron will be degraded and
the snRNPs used again
U2
A
Intron plus U2,
U5, and U6
U6
5′
Figure 12.22
Exon 1
U5
Exon 2
3′
Two connected
exons
55
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Show view). You may see blank slides
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in Presentation Mode and playing each
animation. Most animations will require
the latest version of the Flash Player,
which is available at
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56
Intron Advantage?

One benefit of genes with introns is a phenomenon
called alternative splicing

A pre-mRNA with multiple introns can be spliced in
different ways


This will generate mature mRNAs with different
combinations of exons
This variation in splicing can occur in different cell
types or during different stages of development
57
Intron Advantage?

The biological advantage of alternative splicing is
that two (or more) polypeptides can be derived
from a single gene

This allows an organism to carry fewer genes in its
genome
58
Alternative Splicing


One very important biological advantage of introns in
eukaryotes is the phenomenon of alternative splicing
Alternative splicing refers to the phenomenon that
pre-mRNA can be spliced in more than one way



Alternatively splicing produces two or more polypeptides
with different amino acid sequences
In most cases, large sections of the coding regions are the
same, resulting in alternative versions of a protein that
have similar functions
Nevertheless, there will be enough differences in amino
acid sequences to provide each polypeptide with its own
unique characteristics
59
Alternative Splicing

The degree of splicing and alternative splicing
varies greatly among different species

Baker’s yeast contains about 6,300 genes

~ 300 (i.e., 5%) encode mRNAs that are spliced


Only a few of these 300 have been shown to be alternatively spliced
Humans contain ~ 25,000 genes

Most of these encode mRNAs that are spliced


It is estimated that about 70% are alternatively spliced
Note: Certain mRNAs can be alternatively spliced to produce dozens
of different mRNAs
60
Alternative Splicing

Figure 15.19 considers an example of alternative
splicing for a gene that encodes a-tropomyosin


This protein functions in the regulation of cell contraction
It is found in




Smooth muscle cells (uterus and small intestine)
Striated muscle cells (cardiac and skeletal muscle)
Also in many types of nonmuscle cells at low levels
The different cells of a multicellular organism regulate
contractibility in subtly different ways

One way to accomplish this is to produce different forms of
a-tropomyosin by alternative splicing
61
Found in the mature mRNA
from all cell types
Not found in all
mature mRNAs
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Intron
5′
1
2
α-tropomyosin pre-mRNA
Exon
3
4
5
6
7
8
9
10
11
12
13 14
3′
Constitutive exons
Alternative
splicing
5′
1
2
4
5
6
Alternative exons
3′
8
9
10 14
8
9
10 11 12
Smooth muscle cells
or
5′
1
3
4
5
6
3′
Striated muscle cells
These alternatively spliced versions of a-tropomyosin vary in
function to meet the needs of the cell type in which they are found
Figure 15.19
Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
62
Capping

Most mature mRNAs have a 7-methylguanosine
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 12.23, capping is a three-step
process
63
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5′
O
O
O
O– P O P O P O CH2
O–
O–
O–
HH
O
Base
O
HH
O
OH
O P OO CH2
O–
P
O
O
O–
O
P
O
O–
P
Base
O
CH2
O
O–
H
O
H
H
O
OH
P
O–
O
CH2
H
Rest of mRNA
3′
RNA 5′-triphosphatase
removes a phosphate.
Pi
O
O–
O
P
O
O–
P
Base
O
CH2
O
O–
H
O
H
H
O
OH
P
O–
O
CH2
H
Rest of mRNA
Figure 12.23
PPi
Guanylyltransferase
hydrolyzes GTP. The GMP is
attached to the 5′ end, and
PPi is released.
64
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
H
O
N
NH2
N
N
N
OH
H
O
H
HO
CH2
O
H
O
O–
H
O
P
O
O
P
O
O–
P
Base
O
CH2
O–
H
O
H
O
CH3
N
OH
O
OH
P
O–
O
CH2
H
H
O
H
HO
H
O
H
Methyltransferase attaches
a methyl group.
N+
N
H
Rest of mRNA
N
NH2
O
H
CH2
O
P
O–
O
O
P
O–
O
O
P
Base
O
CH2
O–
H
7-methylguanosine cap
O
O
H
H
O
OH
P
O–
O
CH2
H
Rest of mRNA
Figure 12.23
65
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
66
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 12.24
67
Figure 12.24
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Polyadenylation signal sequence
5′
Consensus sequence in
higher eukaryotes
5′
3′
AAUAAA
Endonuclease cleavage occurs
about 20 nucleotides downstream
from the AAUAAA sequence.
AAUAAA
PolyA-polymerase adds
adenine nucleotides
to the 3′ end.
5′
AAUAAA
Appears to be important in the
stability of mRNA and the
translation of the polypeptide
AAAAAAAAAAAA.... 3′
PolyA tail
Length varies between species
From a few dozen adenines
to several hundred
68
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