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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
3
Words to Know
Court reporter transcribing court
testimony
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
5
1. Check out
2. Make many copies of the same page
3. Return unaltered
4. Distribute and incite a riot!
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
Gene Expression
6
Figure 12.1
The central dogma of geneticsmakes DNA copies that are transmittedfrom cell to cell and from parent tooffspring.
DNA replication:
produces an RNA copy of a gene.
Chromosomal DNA: stores information in units called genes.
Transcription:
produces a polypeptide using theinformation in mRNA.
Translation:
Gene
Polypeptide: becomes part of a functional protein that contributes to an organism's traits.
Messenger RNA: a temporary copy of a gene that contains information to make a polypeptide.
7
8
Is this simplistic?
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
Transcription occurs in three stages Initiation Elongation Termination
These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA
sequences
The Stages of Transcription
10
DNA of a gene
Promoter Terminator
Completed RNAtranscript
RNApolymerase
5′ end of growingRNA transcript
Open complex
Initiation: The promoter functions as a recognitionsite for transcription factors (not shown). The transcriptionfactor(s) enables RNA polymerase to bind to the promoter.Following binding, the DNA is denatured into a bubbleknown as the open complex.
Elongation/synthesis of the RNA transcript:RNA polymerase slides along the DNAin an open complex to synthesize RNA.
Termination: A terminator is reached that causes RNApolymerase and the RNA transcript to dissociate fromthe DNA.
RNA polymerase
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 12.3
Transcription
11
12
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Once they are made, RNA transcripts play different functional roles Refer to Table 12.1
Well over 90% of all genes are structural genes which are transcribed into mRNA Final functional products are polypeptides
The other RNA molecules in Table 12.1 are never translated Final functional products are RNA molecules
RNA Transcripts Have Different Functions
13
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
RNA Transcripts Have Different Functions
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 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
Promoters
17
Template strand
Transcription
Coding strand Transcriptionalstart site
16 –18 bp+1
–35 sequence –10 sequence
Promoter region
G T
C ATA
CG
AT
AT
TA
TA
TA
TA
AT
AT
AT
3′ 5′
5′
5′ 3′
3′
RNAA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 12.4 The conventional numbering system of promoters
Bases preceding the start site are
numbered in a negative direction
There is no base numbered 0
Bases to the right are numbered in a
positive direction
Most of the promoter region is labeled with negative numbers
18
Figure 12.4 The conventional numbering system of promoters
The promoter may span a large region, but specific short sequence elements are
particularly critical for promoter recognition and activity level
Sometimes termed the Pribnow box, after its
discoverer
Sequence elements that play a key role in transcription
Template strand
Transcription
Coding strand Transcriptionalstart site
16 –18 bp+1
–35 sequence –10 sequence
Promoter region
G T
C ATA
CG
AT
AT
TA
TA
TA
TA
AT
AT
AT
3′ 5′
5′
5′ 3′
3′
RNAA
19
RNA polymerase is the enzyme that catalyzes the synthesis of RNA
In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme
Five subunits = a2bb’ Sigma factor
One subunit = s
These subunits play distinct functional roles
Initiation of Bacterial Transcription
20
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
Initiation of Bacterial Transcription
21
Figure 12.6
Binding of factor protein to DNA double helix
22
Amino acids within the a helices hydrogen
bond with bases in the -35 and -10 promoter
sequences
Turn
α helicesbinding to themajor groove
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
HELIX
HELIX
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
Figure 12.7
–10
–35
–35
–35
–35
–10
–10
–10
RNA polymerase
RNA polymeraseholoenzyme
After sliding along the DNA, σfactor recognizes a promoter, andRNA polymerase holoenzymeforms a closed complex.
An open complex is formed, anda short RNA is made.
σ factor is released, and thecore enzyme is able to proceeddown the DNA.
σ factor
σ factor
RNA transcript
Open complex
Closed complex
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Promotor region
RNA polymerasecore enzyme
24
25
In class skit
Characters:Character Played By
A shy female college student
A cute dude
A helpful friend Dr. Ballard
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
Elongation in Bacterial Transcription
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
Which is the template strand?Which is the coding strand?
29
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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
Elongation in Bacterial Transcription
30
Similar to the synthesis of DNA
via DNA polymerase
Figure 12.8
Key points:
• 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).
• The complementarity rule is the same as the AT/GC rule except that U is substituted for T in the RNA.
3′
5′
5′
3′
3′
5′
RNA polymerase
Direction oftranscription
Rewinding of DNA
RNA
Open complex
Codingstrand
Template strand
Unwinding of DNA
Nucleotide beingadded to the 3′end of the RNA
RNA–DNAhybridregion
Templatestrand
C G
GT
T
A
AG C
CA U
Codingstrand
Nucleosidetriphosphates
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.s
31
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
Termination of Bacterial Transcription
32
r-dependent terminationFigure 12.10
5′
5′
5′
3′
5′
Terminator
rut
RNA polymerase reaches theterminator. A stem-loopcauses RNA polymeraseto pause.
Stem-loop
Terminator
RNA polymerase pausesdue to its interaction withthe stem-loop structure. ρprotein catches up to the opencomplex and separates theRNA-DNA hybrid.
3′
3′
3′
ρ recognition site (rut)
ρ recognitionsite in RNA
ρ protein binds to therut site in RNA and movestoward the 3′ end.
ρ protein
Rho protein is a helicase
rho utilization site
33
Stem-loop that causesRNA polymerase to pause
U-rich RNA inthe RNA-DNA hybrid
5′
5′3′
While RNA polymerase pauses,the U-rich sequence is not able tohold the RNA-DNA hybrid together.Termination occurs.
NusA
Terminator
UU
UU
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• 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
r-independent terminationFigure 12.11
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
Stabilizes the RNA pol
pausing
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
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
Eukaryotic RNA Polymerases
36
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
Sequences of Eukaryotic Structural Genes
37
TATA box
Core promoter
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences: TATAAA
Common location forregulatory elements suchas GC and CAAT boxes
–100 –50 –25 +1
Py2CAPy5
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Usually an adenine
• 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
Figure 12.13
38
• 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
TATA box
Core promoter
Transcription
Transcriptionalstart site
DNA
Coding-strand sequences: TATAAA
Common location forregulatory elements suchas GC and CAAT boxes
–100 –50 –25 +1
Py2CAPy5
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 12.13
39
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
Sequences of Eukaryotic Structural Genes
40
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
RNA Polymerase II and its Transcription Factors
41
TFIID TFIIB
TFIID TFIIB
TFIID TFIIB
TFIID
TFIIF
TATA box
TFIID binds to the TATA box. TFIID isa complex of proteins that includes theTATA-binding protein (TBP) and severalTBP-associated factors (TAFs).
TFIIB binds to TFIID.
TFIIB acts as a bridge to bindRNA polymerase II and TFIIF.
TFIIE and TFIIH bind to RNApolymerase II to form a preinitiationor closed complex.
TFIIH acts as a helicase to form anopen complex. TFIIH also phosphorylatesthe CTD domain of RNA polymerase II.CTD phosphorylation breaks the contactbetween TFIIB and RNA polymerase II.TFIIB, TFIIE, and TFIIH are released.
RNA polymerase II
Preinitiation complex
Open complex
CTD domain ofRNA polymerase II
PO4
TFIIF
TFIIE
TFIID
TFIIB
TFIIF
TFIIETFIIH
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PO4
TFIIH
Figure 12.14
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
A closed complex
RNA pol II can now proceed to the
elongation stageReleased after the open complex is
formed
42
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
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
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
Splicing
48
49
Figure 12.20
In eukaryotes, the transcription of structural genes produces a long transcript known as pre-mRNA
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)
HH
OOH
H
CH2O
O
P
P
A
Exon 1
Intron
Exon 2
Spliceosome
HH
OO
H
CH2O
O
P
PP
A
3′OH
3′
3′
HH
O
H
CH2O
O
P
P
A
PO
3′5′
5′
5′
mRNA
(c) Pre-mRNA
2′
2′
2
2′
2′
2
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
50
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
Pre-mRNA Splicing
51
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
Pre-mRNA Splicing
52
Figure 12.21
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
Sequences shown in bold are highly conserved
Corresponds to the boxed adenine in Figure 12.22
Serve as recognition sites for the binding of the spliceosome
The pre-mRNA splicing mechanism is shown in Figure 12.22
3′5′
3′ splice siteBranch site
IntronExon ExonCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
UACUUAUCC Py12N Py AGGA/CGGU Pu AGUA
5′ splice site
53
U1
3′5′
5′ splice site 3′ splice siteBranch site
AGU
Exon 1 Exon 2
U1 binds to 5′ splice site.U2 binds to branch site.
AG
3′5′A
U4/U6 and U5 trimer binds. Intronloops out and exons are broughtcloser together.
U1 snRNP U2 snRNP
3′5′
A
U5 snRNP
U4/U6 snRNP
U2
Intron loops out and exons brought closer
together
Figure 12.22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
54
Figure 12.22
Intron will be degraded and the snRNPs used again
U1U4
3′5′
3′5′
5′ splice site is cut.5′ end of intron is connected to theA in the branch site to form a lariat.U1 and U4 are released.
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.
A
A
U5U6
U5U6
U2
Intron plus U2,U5, and U6
Two connectedexons
Exon 1 Exon 2
U2
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Cleavage may becatalyzed by snRNAmolecules within U2and U6
55
56
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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
Intron Advantage?
57
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
Intron Advantage?
58
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
Alternative Splicing
59
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
Alternative Splicing
60
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
Alternative Splicing
61
1 654 10987 1413121132
1 654 1098 142
1 654 1098 12113
Intron Exon α-tropomyosin pre-mRNA
5′ 3′
5′ 3′
Constitutive exonsAlternativesplicing
or
Alternative exons
Smooth muscle cells
5′ 3′ Striated muscle cells
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Figure 15.19 Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
Found in the mature mRNA from all cell types
Not found in all mature mRNAs
These alternatively spliced versions of a-tropomyosin vary in function to meet the needs of the cell type in which they are found
62
Most mature mRNAs have a 7-methylguanosine covalently attached at their 5’ end This event is known as capping
Capping occurs as the pre-mRNA is being synthesized by RNA pol II Usually when the transcript is only 20 to 25 bases long
As shown in Figure 12.23, capping is a three-step process
Capping
63
Figure 12.23
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
P
HH
OH
HH
Base
OCH2
CH2
OO
OP O-
HH
OH
HH
Base
OOO
OP CH2
CH2
O–
OP
O–
OOP
O–
Rest of mRNA
HH
OH
HH
Base
OCH2
CH2
Rest of mRNA
RNA 5′-triphosphataseremoves a phosphate.
Guanylyltransferasehydrolyzes GTP. The GMP isattached to the 5′ end, andPPi is released.
PPi
Pi
5′
3′
OO
O
P
O–
O
P
O–
O
O
P
O–
O–
O
O
O
O–
O
O
O
P O–
OO
O
P
O–
O
P
O–
O–
O–
64
Figure 12.23
HH
OH
HH
Base
OCH2
CH2
CH2
Rest of mRNA
HH
OH
HH
Base
OCH2
CH2
CH3
CH2
Rest of mRNA
Methyltransferase attachesa methyl group.
7-methylguanosine cap
H
HH
OH
HO
O
NH2
H
N
N
H
N
O
N
H
HH
OH
HO
O
NH2
H
N
N
H
N
O
N
+
O
O
P
O–
O
O
O
P O–
OO
O
P
O–
O
O P
O–
O
O
P
O–
O
O
O
P O–
OO
O
P
O–
O
O
P
O–
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
65
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
Capping
66
Most mature mRNAs have a string of adenine nucleotides at their 3’ ends This is termed the polyA tail
The polyA tail is not encoded in the gene sequence It is added enzymatically after the gene is completely
transcribed
The attachment of the polyA tail is shown in Figure 12.24
Tailing
67
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′ 3′
5′
5′ 3′
Endonuclease cleavage occursabout 20 nucleotides downstreamfrom the AAUAAA sequence.
PolyA-polymerase addsadenine nucleotidesto the 3′ end.
Polyadenylation signal sequence
AAUAAA
AAUAAA
AAUAAA
PolyA tail
AAAAAAAAAAAA....
Figure 12.24
Consensus sequence in higher eukaryotes
Appears to be important in the stability of mRNA and the
translation of the polypeptide Length varies between species
From a few dozen adenines to several hundred
68