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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
that is transcribed into RNA and has a
cellular function
Some DNA sequences are transcribed into
RNA but the RNA has no known function; for
our purposes, these sequences are not genes
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
Function catalyzed by RNA polymerase
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
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 genetics
makes DNA copies that are transmitted
from cell to cell and from parent to
offspring.
DNA replication:
produces an RNA copy of a gene.
Chromosomal DNA: stores information in
units called genes.
Transcription:
produces a polypeptide using the
information 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
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
phenotype
Or: How the book results 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 RNA
transcript
RNA
polymerase
5 end of growing
RNA transcript
Open complex
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.
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.
RNA polymerase
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Figure 12.3
Transcription
11
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12
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 dont 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 Transcriptional
start site
16 18 bp
+1
35 sequence 10 sequence
Promoter region
G
T
C
A
T
A
C
G
A
T
A
T
T
A
T
A
T
A
T
A
A
T
A
T
A
T
3 5
5
5 3
3
RNA
A
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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 Transcriptional
start site
16 18 bp
+1
35 sequence 10 sequence
Promoter region
G
T
C
A
T
A
C
G
A
T
A
T
T
A
T
A
T
A
T
A
A
T
A
T
A
T
3 5
5
5 3
3
RNA
A
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 =
2

Sigma factor
One subunit =
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
Amino acids within the
helices hydrogen
bond with bases in the
-35 and -10 promoter
sequences
Turn
helices
binding to the
major groove
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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
Figure 12.7
10
35
35
35
35
10
10
10
RNA polymerase
RNA polymerase
holoenzyme
After sliding along the DNA,
factor recognizes a promoter, and
RNA polymerase holoenzyme
forms a closed complex.
An open complex is formed, and
a short RNA is made.
factor is released, and the
core enzyme is able to proceed
down the DNA.
factor
factor
RNA transcript
Open complex
Closed complex
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Promotor region
RNA polymerase
core enzyme
24
Character Played By
A shy female college student
A cute dude
A helpful friend Dr. Ballard
25
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 strands 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 Us for Ts)
27
28
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29
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
Figure 12.8
3
5
5
3
3
5
RNA polymerase
Direction of
transcription
Rewinding of DNA
RNA
Open complex
Coding
strand
Template strand
Unwinding of DNA
Nucleotide being
added to the 3
end of the RNA
RNADNA
hybrid
region
Template
strand
C
G
G
T
T
A
A
G
C
C
A
U
Coding
strand
Nucleoside
triphosphates
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 (rho)
2. rho-independent termination
Does not require
Termination of Bacterial
Transcription
32
-dependent termination
Figure 12.10
5
5
5
3
5
Terminator
rut
RNA polymerase reaches the
terminator. A stem-loop
causes RNA polymerase
to pause.
Stem-loop
Terminator
RNA polymerase pauses
due to its interaction with
the stem-loop structure.
protein catches up to the open
complex and separates the
RNA-DNA hybrid.
3
3
3
recognition site (rut)
recognition
site in RNA
protein binds to the
rut site in RNA and moves
toward the 3 end.
protein
Rho protein is
a helicase
rho utilization
site
33
Stem-loop that causes
RNA polymerase to pause
U-rich RNA in
the RNA-DNA
hybrid
5
5
3
While RNA polymerase pauses,
the U-rich sequence is not able to
hold the RNA-DNA hybrid together.
Termination occurs.
NusA
Terminator
U
U
U
U
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-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
-independent termination
Figure 12.11
U
RNA
-A
DNA
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 rRNAgenes (except for the 5S rRNA)
RNA pol II
Transcribes all structural genes
Thus, synthesizes all mRNAs
Transcribes some snRNAgenes
RNA pol III
Transcribes all tRNAgene
And the 5S rRNAgene
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
Transcriptional
start site
DNA
Coding-strand sequences: TATAAA
Common location for
regulatory elements such
as GC and CAAT boxes
100 50 25 +1
Py
2
CAPy
5
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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 termedbasal 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
Transcriptional
start site
DNA
Coding-strand sequences: TATAAA
Common location for
regulatory elements such
as GC and CAAT boxes
100 50 25 +1
Py
2
CAPy5
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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 wont 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
TFIID
TFIID TFIIB
TFIID
TFIIF
TATA box
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).
TFIIB binds to TFIID.
TFIIB acts as a bridge to bind
RNA polymerase II and TFIIF.
TFIIE and TFIIH bind to RNA
polymerase II to form a preinitiation
or closed complex.
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.
RNA polymerase II
Preinitiation complex
Open complex
CTD domain of
RNA polymerase II
PO
4
TFIIF
TFIID
TFIIB
TFIIF
TFIIE
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PO
4
Figure 12.14
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A closed complex
RNA pol II can now
proceed to the
elongation stage
Released after the
open complex is
formed
You dont 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
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 wont 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 (well 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)
H H
O
OH
H
CH2 O
O
P
P
A
Exon 1
Intron
Exon 2
Spliceosome
H H
O
O
H
CH2 O
O
P
P
P
A
3
OH
3
3
H H
O
H
CH2 O
O
P
P
A
P
O
3 5
5
5
mRNA
(c) Pre-mRNA
2
2
2
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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 site Branch site
Intron Exon Exon
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UACUUAUCC
Py
12
N Py AGG A
/
C
GGU Pu AGUA
5 splice site
53
U1
3 5
5 splice site 3 splice site
Branch site
A
GU
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. Intron
loops out and exons are brought
closer 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
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54
Figure 12.22
Intron will be degraded and
the snRNPs used again
U1
U4
3
5
3 5
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.
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
U5
U6
U5 U6
U2
Intron plus U2,
U5, and U6
Two connected
exons
Exon 1
Exon 2
U2
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Cleavage may be
catalyzed by snRNA
molecules within U2
and U6
55
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56
Experiment 12A: Identification of
Introns Via Microscopy
In the late 1970s, several research groups
investigated the presence of introns in eukaryotic
structural genes
One of these groups was led by Phillip Leder
Leder used electron microscopy to identify introns in
the -globin gene
Leder used a strategy that involved hybridization
Experiment 12A: Identification of
Introns Via Microscopy
Double-stranded DNA of the cloned -globin gene
is first denatured
Then mixed with mature -globin mRNA
The mRNA is complementary to the template
strand of the DNA
So the two will bind or hybridize to each other
If the DNA is allowed to renature, this complex will prevent the
reformation of the DNA double helix
Refer to Figure 12.18
(a) No introns in the DNA
+
DNA region complementary to mRNA
R loop
mRNA
Discontinuous regions of DNA
that are complementary to mRNA
Mature mRNA
Pre-mRNA
Splicing
Transcription
mRNA bound to DNA
Template
strand
Coding
strand
Template
strand
mRNA bound to DNA
mRNA bound to template strand
R loop R loop
Intron DNA
The coding strand then
binds to the template
strand, but it loops out
where the RNA is
already bound.
Intron DNA of
template strand
Intron
5 5
5
3
3
5
3
5
5 3
3
3
5 3
5
5
3
3
5
3
Mix together denatured DNA and mature mRNA.
The mature mRNA binds to template strand, which
causes the intron DNA to loop out.
(b) One intron in the DNA. The intron in the pre-mRNA is spliced out.
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Figure 12.18
RNA displacement
loop
mRNA cannot hybridize here
Because the intron has been
spliced out of the mRNA
The Hypothesis
The -globin gene from the mouse contains one
or more introns
Testing the Hypothesis
Refer to Figure 12.19
Figure 12.19
Mix together the -globin mRNA and
cloned DNA of the -globin gene.
IsolatematuremRNA for themouse
-globin gene. Note: GlobinmRNA is
abundant inreticulocytes, whichare
immaturered blood cells.
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Experimental level Conceptual level
1.
2.
Separatethedouble-stranded DNA and
allow themRNA to hybridize. This is
doneusing 70% formamide, at 52C, for
16 hours.
3.
Dilutethesampleto decreasethe
formamideconcentration. This allows
theDNA to re-formadouble-stranded
structure. Note: TheDNA cannot form
adouble-stranded structureinregions
wherethemRNA has already hybridized.
4.
Spread thesampleonto amicroscopy
grid.
5.
Stainwithuranyl acetateand shadow
withheavy metal. Note: Thetechnique
of electronmicroscopy is described in
theAppendix.
6.
View thesampleunder theelectron
microscope.
7.
70%
formamide
Incubator
Platinum
electrode
Platinum
atoms
Vacuumevaporator
Specimen
mRNA
Solution
of cloned
-globin
DNA
-globin DNA
Solution of
-globin
mRNA
Cloned
-globin
DNA
Interpreting the Data
12-58
Hybridization caused the
formation of two R loops,
separated by a double-
stranded DNA region
This suggests that the -globin
gene contains introns
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R loop
Intron
R loop
. . . 1978, . 75. . 2, . 727.
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?
63
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?
64
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
65
The degree of splicing and alternative splicing
varies greatly among different species
Bakers 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
66
Figure 15.19 considers an example of alternative
splicing for a gene that encodes -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
-tropomyosin by alternative splicing
Alternative Splicing
67
1 6 5 4 10 9 8 7 14 13 12 11 3 2
1 6 5 4 10 9 8 14 2
1 6 5 4 10 9 8 12 11 3
Intron
Exon
-tropomyosin pre-mRNA
5 3
5 3
Constitutive exons
Alternative
splicing
or
Alternative exons
Smooth muscle cells
5 3 Striated muscle cells
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Figure 15.19 Alternative ways that the rat -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 -tropomyosin vary in
function to meet the needs of the cell type in which they are found
68
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
69
Figure 12.23
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
P
H H
OH
H H
Base
O
CH
2
CH
2
O
O
O
P O
-
H H
OH
H H
Base
O
O O
O
P CH2
CH2
O

O
P
O

O
O
P
O

Rest of mRNA
H H
OH
H H
Base
O
CH
2
CH
2
Rest of mRNA
RNA 5-triphosphatase
removes a phosphate.
Guanylyltransferase
hydrolyzes GTP. The GMP is
attached to the 5 end, and
PP
i
is released.
PP
i
P
i
5
3
O O
O
P
O

O
P
O

O
O
P
O

O
O
O
O

O
O
O
P O

O O
O
P
O

O
P
O

70
Figure 12.23
H H
OH
H H
Base
O
CH
2
CH
2
CH
2
Rest of mRNA
H H
OH
H H
Base
O
CH
2
CH
2
CH
3
CH
2
Rest of mRNA
Methyltransferase attaches
a methyl group.
7-methylguanosine cap
H
H
H
OH
HO
O
NH
2
H
N
N
H
N
O
N
H
H
H
OH
HO
O
NH
2
H
N
N
H
N
O
N
+
O
O
P
O

O
O
O
P O

O O
O
P
O

O
O P
O

O
O
P
O

O
O
O
P O

O O
O
P
O

O
O
P
O

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
71
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
72
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
73
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5
3
5
5
3
Endonuclease cleavage occurs
about 20 nucleotides downstream
from the AAUAAA sequence.
PolyA-polymerase adds
adenine nucleotides
to 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
74

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