*See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes.
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–   either an RNA or a polypeptide
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12-2

•   Transcription literally means the act or process of making a copy
•   In genetics, the term refers to the copying of a
DNA sequence into an RNA sequence
•   The structure of DNA is not altered as a result of this process
–   It can continue to store information
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12-3

n
Structural genes encode the amino acid sequence of a polypeptide n   n   n
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 n
This path from gene to trait is called the central dogma of genetics n
Refer to Figure 12.1
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12-4

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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.
Figure 12.1
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.
12-5

•   A key concept is that DNA base sequences define the beginning and end of a gene and regulate the level of
RNA synthesis
•   Another important concept is that proteins must recognize and act on DNA for transcription to occur
•   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
•   Figure 12.2 shows common organization of a bacterial gene
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12-6

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Regulatory " sequence
DNA mRNA 5 ′
Promoter
Start " codon
Ribosome " binding site
Transcription
Terminator
DNA:
• Regulatory sequences: site for the binding of regulatory "
proteins; the role of regulatory proteins is to influence "
the rate of transcription. Regulatory sequences can be "
found in a variety of locations.
• Promoter: site for RNA polymerase binding; signals "
the beginning of transcription.
• Terminator: signals the end of transcription.
Many codons
Stop "
Signals the end of protein synthesis
3 codon
′ mRNA:
• Ribosome-binding site: site for ribosome binding; "
translation begins near this site in the mRNA. In eukaryotes, "
the ribosome scans the mRNA for a start codon.
• Start codon: specifies the first amino acid in a polypeptide "
sequence, usually a formylmethionine (in bacteria) or a "
methionine (in eukaryotes).
• Codons: 3-nucleotide sequences within the mRNA that "
specify particular amino acids. The sequence of codons "
within mRNA determines the sequence of amino acids "
within a polypeptide.
• Stop codon: specifies the end of polypeptide synthesis.
• Bacterial mRNA may be polycistronic, which means it "
encodes two or more polypeptides.
Figure 12.2
12-7

n   n   n   n
The DNA strand that is actually transcribed (used as the template) is termed the template strand n
The RNA transcript is complementary to the template strand
The opposite strand is called the coding strand or the sense strand as well as the nontemplate strand n
The base sequence is identical to the RNA transcript n
Except for the substitution of uracil in RNA for thymine in DNA
Transcription factors recognize the promoter and regulatory sequences to control transcription mRNA sequences such as the ribosomal-binding site and codons direct translation
12-8
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n
Transcription occurs in three stages n
Initiation n   n
Elongation
Termination n
These steps involve protein-DNA interactions n
Proteins such as RNA polymerase interact with DNA sequences
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12-9

Promoter
5 ′ end of growing "
RNA transcript
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DNA of a gene
Transcription !
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.
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
12-10

n   n   n
Once they are made, RNA transcripts play different functional roles n
Refer to Table 12.1
Well over 90% of all genes are structural genes which are transcribed into mRNA n
Final functional products are polypeptides
The other RNA molecules in Table 12.1 are never translated n
Final functional products are RNA molecules
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12-11

n
The RNA transcripts from nonstructural genes are not translated n   n   n
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 n
For example n
Ribosomes n   n
Spliceosomes
Signal recognition particles
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12-12

12-13

•   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
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12-14

n   n
Promoters are DNA sequences that “ promote ” gene expression n
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 n
The bases in a promoter sequence are numbered in relation to the transcription start site n
Refer to Figure 12.4
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12-15

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
Promoter region
–35 sequence 16 –18 bp –10 sequence
5 ′
Transcriptional start site
+1
3 ′
"
T
A
T
A
G A
C T
C
G
A
T
T
A
A
T
T
A
A
T
A
T
T
A
A
T
3 ′ 5 ′
Template strand
5 ′
A
RNA
3 ′
Bases to the right are numbered in a positive direction
Transcription
Figure 12.4 The conventional numbering system of promoters
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12-16

Sequence elements that play a key role in transcription
The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level
Transcriptional " start site
Coding strand
Promoter region
–35 sequence 16 –18 bp –10 sequence
5 ′
T
A
T
A
G A
C T
C
G
A
T
T
A
A
T
T
A
A
T
A
T
T
A
3 ′
+1
A
T
3
5 ′
′
Template strand
Sometimes termed the
Pribnow box, after its discoverer
5 ′
A
RNA
3 ′
Transcription
Figure 12.4 The conventional numbering system of promoters
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12-17

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
–35 region –10 region +1 Transcribed lac operon
For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences of the -35 and -10 regions lac I trp operon rrn X rec A lex A
The most commonly occurring bases tRNA tyr
TTTACA N
17
TATGTT
N
6
A
GCGCAA N
17
CATGAT
N
7
A
TTGACA N
17
TTAACT
N
7
A
TTGTCT N
16
TAATAT
N
7
A
TTGATA N
16
TATAAT
N
7
A
TTCCAA N
17
TATACT
N
7
A
TTTACA N
16
TATGAT
N
7
A
Consensus TTGACA TATAAT
Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters
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12-18

n
RNA polymerase is the enzyme that catalyzes the synthesis of RNA n
In E. coli , the RNA polymerase holoenzyme is composed of n   n
Core enzyme n
Five subunits = α
2
ββ ʼ ω
Sigma factor n
One subunit = σ n
These subunits play distinct functional roles
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12-19

n
The RNA polymerase holoenzyme binds loosely to the DNA n
It then scans along the DNA, until it encounters a promoter region n
When it does, the sigma factor recognizes both the –35 and –10 regions n
A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA n
Refer to Figure 12.6
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12-20

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Amino acids within the
α
helices hydrogen bond with bases in the
-35 and -10 promoter sequences
α helices " binding to the " major groove
"
Turn
Figure 12.6
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12-21

n
The binding of the RNA polymerase to the promoter forms the closed complex n
Then, the open complex is formed when the
TATAAT box in the -10 region is unwound n
A short RNA strand is made within the open complex n
The sigma factor is released at this point n
This marks the end of initiation n
The core enzyme now slides down the DNA to synthesize an RNA strand n
This is known as the elongation phase
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12-22

Figure 12.7
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RNA polymerase
Promotor region
σ factor
–35 –10
RNA polymerase " holoenzyme
After sliding along the DNA, σ" factor recognizes a promoter, and "
RNA polymerase holoenzyme " forms a closed complex.
–35
–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.
–35
RNA polymerase " core enzyme
–10
σ factor
RNA transcript
12-23

n
The RNA transcript is synthesized during the elongation stage n
The DNA strand used as a template for RNA synthesis is termed the template or antisense strand n
The opposite DNA strand is called the coding strand n
It has the same base sequence as the RNA transcript n
Except that T in DNA corresponds to U in RNA
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12-24

n
The open complex formed by the action of RNA polymerase is about 17 bases long n
Behind the open complex, the DNA rewinds back into a double helix n
On average, the rate of RNA synthesis is about 43 nucleotides per second! n
Figure 12.8 depicts the key points in the synthesis of an RNA transcript
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12-25

Similar to the synthesis of DNA via DNA polymerase
Figure 12.8
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Coding " strand
Template " strand
5 ′
RNA
5 ′ 3 ′
A
U
G
C
T
A
C
G
G
T
C
A
Template strand
3 ′
Rewinding of DNA
RNA polymerase
Open complex
Unwinding of DNA
Direction of " transcription
Coding " strand
3 ′
RNA–DNA " hybrid " region
5 ′
Nucleotide being " added to the 3 ′" end of the RNA
Nucleoside " triphosphates 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.
12-26

n
Termination is the end of RNA synthesis n
It occurs when the short RNA-DNA hybrid of the open complex is forced to separate n
This releases the newly made RNA as well as the RNA polymerase n
E. coli has two different mechanisms for termination n   n
1. rho-dependent termination n
Requires a protein known as
ρ
(rho)
2.
rho-independent termination n
Does not require ρ
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12-27

r ho ut ilization site
Rho protein is a helicase
5 ′
ρ recognition site (rut)
Terminator rut
3 ′
ρ recognition " site in RNA
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
Stem-loop
3 ′
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 ′
Figure 12.10
5 ′
ρ
-dependent termination
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12-28

•   ρ -independent termination is facilitated by two sequences in the RNA
–   1. A uracil-rich sequence located at the 3 ʼ end of the RNA
–   2. A stem-loop structure upstream of the uracil-rich sequence
U
RNA
-A
DNA
hydrogen bonds are relatively weak
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5 ′
U-rich RNA in " the RNA-DNA "
hybrid
Stem-loop that causes "
RNA polymerase to pause NusA
While RNA polymerase pauses, " the U-rich sequence is not able to " hold the RNA-DNA hybrid together.
"
Termination occurs.
Terminator
Stabilizes the RNA pol pausing
No protein is required to physically remove the RNA from the DNA
This type of termination is also called intrinsic
5 ′
U
U
U
U
3 ′
Figure 12.11 ρ
-independent termination
12-29

•   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
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12-30

n
Nuclear DNA is transcribed by three different RNA polymerases n   n   n
RNA pol I n
Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II n   n
Transcribes all structural genes n
Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III n   n
Transcribes all tRNA genes
And the 5S rRNA gene
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12-31

n
All three are very similar structurally and are composed of many subunits n
There is also a remarkable similarity between the bacterial RNA pol and its eukaryotic counterparts n
Refer to Figure 12.13
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12-32

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Structure of
RNA polymerase
© From Seth Darst, Bacterial RNA polymerase.
Current Opinion in Structural Biology.
Reprinted with permission of the author.
(a) Structure of a bacterial !
RNA polymerase
© From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of
Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution." Science, Vol.
292:5523, 1863-1876, June 8, 2001.
Structure of a eukaryotic !
RNA polymerase II (yeast)
5 ′ 3 ′
Transcribed DNA "
(upstream)
Figure 12.12
Exit
Lid
5 ′
Clamp
Rudder
Entering DNA "
(downstream)
3 ′
Wall
Mg 2+
Bridge
Jaw
Catalytic " site
NTPs enter " through a pore
Transcription
(b) Schematic structure of RNA polymerase
5 ′
12-33

n
Eukaryotic promoter sequences are more variable and often more complex than those of bacteria n
For structural genes, at least three features are found in most promoters n
Regulatory elements n
TATA box n
Transcriptional start site n
Refer to Figure 12.13
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12-34

Figure 12.13
–100
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Core promoter
TATA box
Transcriptional " start site
Coding-strand sequences: TATAAA
Common location for " regulatory elements such " as GC and CAAT boxes
–50 –25
Py
2
CAPy
5
+1
DNA Transcription
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
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12-35

Figure 12.13
–100
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Core promoter
TATA box
Transcriptional " start site
Coding-strand sequences: TATAAA
Common location for " regulatory elements such " as GC and CAAT boxes
–50 –25
Py
2
CAPy5
+1
DNA 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
–   They 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
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12-36

n
Factors that control gene expression can be divided into two types, based on their “ location ” n   cis -acting elements n   n
DNA sequences that exert their effect only over a particular gene
Example: TATA box, enhancers and silencers n   trans -acting elements n
Regulatory proteins that bind to such DNA sequences
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12-37

n
Three categories of proteins are required for basal transcription to occur at the promoter n   n   n
RNA polymerase II
Five different proteins called general transcription factors
(GTFs)
A protein complex called mediator n
Figure 12.14 shows the assembly of transcription factors and RNA polymerase II at the TATA box
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12-38

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
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.
TFIID
Preinitiation complex
TFIIF
A closed complex
Released after the open complex is formed
TFIIB
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.
TFIID
TFIIF
Open complex
RNA pol II can now proceed to the elongation stage
TFIIE
TFIIH
PO
4
PO
4
CTD domain of "
RNA polymerase II
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12-39

n
Basal transcription apparatus n
RNA pol II + the five GTFs n
The third component required for transcription is a large protein complex termed mediator n
It mediates interactions between RNA pol II and various regulatory transcription factors n
Its subunit composition is complex and variable n
Mediator may phosphorylate the CTD of RNA polymerase II and it may regulate the ability of TFIIH to phosphorylate the CTD n
Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation
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12-40

12-41

ʼ
–   Further research is needed to determine if either, or both are correct
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12-42

Possible mechanisms for Pol II termination
RNA polymerase II transcribes a gene " past the polyA signal sequence.
"
5 ′"
3 ′"
PolyA signal sequence "
The RNA is cleaved just past the " polyA signal sequence. RNA " polymerase continues transcribing " the DNA.
"
5 ′"
3 ′"
3 ′"
3 ′"
5 ′"
Allosteric model: After passing the polyA signal sequence, "
RNA polymerase II is destabilized due to the release of " elongation factors or the binding of termination factors (not " shown). Termination occurs.
"
3 ′" 5 ′"
3 ′"
5 ′"
Torpedo model: An exonuclease " binds to the 5 ′ end of the RNA " that is still being transcribed and " degrades it in a 5 ′ to 3 ′ direction.
"
3 ′"
Exonuclease catches " up to RNA polymerase II " and causes termination.
"
Exonuclease " 3 ′"
Figure 12.15 5 ′"
3 ′"
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12-43

•   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
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12-44

•   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
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12-45

•   Aside from splicing, RNA transcripts can be modified in several ways
–   For example
•   Trimming of rRNA and tRNA transcripts
•   5 ʼ Capping and 3 ʼ   polyA tailing of mRNA transcripts
–   Refer to Table 12.3
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12-46

12-47

n
Many nonstructural genes are initially transcribed as a large RNA n
This large RNA transcript is enzymatically cleaved into smaller functional pieces n
Figure 12.16 shows the processing of mammalian ribosomal RNA
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12-48

Promoter
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18S 5.8S
28S
Transcription
5 ′
45S rRNA " transcript
This processing occurs in the nucleolus
18S
18S rRNA
5.8S
28S
Cleavage "
(the light pink regions " are degraded)
5.8S
rRNA
28S rRNA
Functional RNAs that are key in ribosome structure
Figure 12.16
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3 ′
12-49

n
Transfer RNAs are also made as large precursors n
These have to be cleaved at both the 5 ʼ and 3 ʼ ends to produce mature, functional tRNAs n
Cleaved by exonuclease and endonuclease n   n   exonucleases cleave a covalent bond between two nucleotides at one end of a strand endonucleases can cleave bonds within a strand n
Figure 12.17 shows the trimming of a precursor tRNA n
Interestingly, the cleavage occurs differently at the 5 ʼ end and the 3 ʼ end
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12-50

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5 ′
Endonuclease "
A
C
C
Exonuclease "
(RNaseD)
3 ′
Endonuclease "
(RNaseP) m G
T
T
P
Covalently modified bases
Found to contain both RNA and protein subunits
However, RNA contains the catalytic activity
Therefore, it is a ribozyme
Figure 12.17
P
IP
Anticodon m G = Methylguanosine
P = Pseudouridine
T = 4-Thiouridine
IP = 2-Isopentenyladenosine
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12-51

n
Three different splicing mechanisms have been identified n   n   n
Group I intron splicing
Group II intron splicing
Spliceosome n
All three cases involve n
Removal of the intron RNA n
Linkage of the exon RNA by a phosphodiester bond
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12-59

n
Splicing among group I and II introns is termed self-splicing n   n
Splicing does not require the aid of enzymes
Instead the RNA itself functions as its own ribozyme n
Group I and II differ in the way that the intron is removed and the exons connected n
Refer to Figure 12.20 n
Group I and II self-splicing can occur in vitro without the additional proteins n
However, in vivo, proteins known as maturases often enhance the rate of splicing
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12-60

CH
2
OH
H
3 ′
OH
O
H
H
OH
Guanosine
G
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Self-splicing introns "
(relatively uncommon)
Intron
G
Guanosine " binding site
Intron
5 ′
Exon 1 G Exon 2
3 ′ 5 ′
Exon 1
A
H
2 ′
OH
O
P
O
H
CH
2
O
H
P
Exon 2
3 ′
Free guanosine bound to site in intron breaks the bond between exon 1 and intron and becomes attached to 5 ʼ end of intron 5 ′
5 ′
G
3 ′
OH
G
3 ′
5 ′
A
P
H
O
2 ′
3 ′
OH
O
H
O
P
CH
2
O
H
P 3 ′
2 ʼ hydroxyl from adenine within intron breaks bond between exon 1 and intron
Results in Exon 1 and 2 covalently joined and free, linear intron
Results in Exon 1 and 2 linkage and intron as lariat
5 ′
G
5 ′"
(a) Group I
RNA
G
3 ′ 5 ′
(b) Group II
A
P
H
O
2 ′
O
H
O
O
P
CH
2
H
P
3 ′
RNA
Figure 12.20
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12-61

n   n   n
In eukaryotes, the transcription of structural genes produces a long transcript known as pre-mRNA
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Intron removed via spliceosome "
(very common in eukaryotes)
Intron
5 ′
Exon 1
A
H
2 ′
O H
O
P
O
H
CH
2
O
H
P
Spliceosome
Exon 2
3 ′
This RNA is altered by splicing and other modifications, before it leaves the nucleus
5 ′
A
P
H
O
2
3 ′
OH
O
O
P
H
CH
2
O
H
P
Splicing in this case requires the aid of a multicomponent structure known as the spliceosome
A
P
H
O
2 ′
O
O
P
CH
2
H
O
H
P
5 ′
Figure 12.20
(c) Pre-mRNA
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3 ′
3 ′
12-62

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12-63

n
The spliceosome is a large complex that splices pre-mRNA n
It is composed of several subunits known as snRNPs (pronounced “ snurps ” ) n
Each snRNP contains s mall n uclear RN A and a set of p roteins
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12-64

n
The subunits of a spliceosome carry out several functions n
1. Bind to an intron sequence and precisely recognize the intron-exon boundaries n
2. Hold the pre-mRNA in the correct configuration n
3. Catalyze the chemical reactions that remove introns and covalently link exons
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12-65

n
Intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries n
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
5 ′
Exon
A /
C
G GU Pu AGUA
5 ′ splice site
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Intron
UACUU A UCC
Branch site
Py
12
N Py AG G
3 ′ splice site
Exon
3 ′
Figure 12.21 Serve as recognition sites for the binding of the spliceosome n
The pre-mRNA splicing mechanism is shown in Figure 12.22
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12-66

5 ′
5 ′
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Exon 1 Exon 2
GU
A AG
5 ′ splice site
Branch site
3 ′ splice site
U1 binds to 5 ′ splice site.
"
U2 binds to branch site.
U1 snRNP U2 snRNP
A
3 ′
3 ′
Intron loops out and exons brought closer together
5 ′
U1
A
U4/U6 and U5 trimer binds. Intron " loops out and exons are brought " closer together.
U2
U4/U6 snRNP
U5 snRNP
3 ′
Figure 12.22
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12-67

Cleavage may be catalyzed by snRNA molecules within U2 and U6
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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.
U1
U4
A
U2
U6 U5
5 ′ 3 ′
Intron will be degraded and the snRNPs used again
5 ′
Exon 1
A
U6
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.
U2
Intron plus U2, "
U5, and U6
U5
Exon 2
3 ′
Two connected " exons
Figure 12.22
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12-68

n
One benefit of genes with introns is a phenomenon called alternative splicing n
A pre-mRNA with multiple introns can be spliced in different ways n
This will generate mature mRNAs with different combinations of exons n
This variation in splicing can occur in different cell types or during different stages of development
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12-69

n
The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene n
This allows an organism to carry fewer genes in its genome
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12-70

n
Most mature mRNAs have a 7-methylguanosine covalently attached at their 5 ʼ end n
This event is known as capping n
Capping occurs as the pre-mRNA is being synthesized by RNA pol II n
Usually when the transcript is only 20 to 25 bases long n
As shown in Figure 12.23, capping is a three-step process
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12-71

Figure 12.23
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O –
O –
P
O
O P
O
O –
O
O
O –
5 ′
P O CH
H
2
O
O
H
O P O -
O
CH
2
OH
H
Base
H
O –
O
P
O –
O
O
P
O –
O
O
P
O –
O
Base
CH
2
O
H
H H
O
O
P
O
O –
CH
2
OH
Rest of mRNA
H
3 ′
RNA 5 ′ -triphosphatase " removes a phosphate.
P i
O –
O
P
O –
O
O
P
O –
O
Base
CH
2
O
H
H H
O
O
P O –
O CH
2
O
Rest of mRNA
H
H
Guanylyltransferase " hydrolyzes GTP. The GMP is " attached to the 5 ′ end, and "
PP i
is released.
PP i
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12-72

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H
N
O
NH
2
N
N
H
H
O
N
OH
H
H
HO CH
2
O
P
O –
O
O O P
O –
O
O
P
O –
O CH
2
H
H
O
H
Base
H
H
N
O
O
P O –
OH
O
O CH
2
Rest of mRNA
NH
2
N
H
H
N
O
OH
N
+
CH
3
H
H
HO
CH
2
O
O
P
O –
O
O
P
O –
Methyltransferase attaches " a methyl group.
O
O
P
O –
O
H
CH
2
H
O
H
Base
H
7-methylguanosine cap
O
P O –
OH
O
O CH
2
Rest of mRNA
Figure 12.23
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12-73

n
The 7-methylguanosine cap structure is recognized by cap-binding proteins n
Cap-binding proteins play roles in the n   n   n
Movement of some RNAs into the cytoplasm
Early stages of translation
Splicing of introns
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12-74

n
Most mature mRNAs have a string of adenine nucleotides at their 3 ʼ ends n
This is termed the polyA tail n
The polyA tail is not encoded in the gene sequence n
It is added enzymatically after the gene is completely transcribed n
The attachment of the polyA tail is shown in
Figure 12.24
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12-75

Figure 12.24
5 ′
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Polyadenylation signal sequence
AAUAAA
Consensus sequence in higher eukaryotes
Endonuclease cleavage occurs " about 20 nucleotides downstream " from the AAUAAA sequence.
3 ′
5 ′
AAUAAA
PolyA-polymerase adds " adenine nucleotides " to the 3 ′ end.
5 ′
AAUAAA
AAAAAAAAAAAA....
3 ′
Appears to be important in the stability of mRNA and the translation of the polypeptide
PolyA tail
Length varies between species
From a few dozen adenines to several hundred
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12-76

n
Change in the nucleotide sequence of an RNA n
Can involve addition or deletion of particular bases n   n
Can also occur through conversion of a base
First discovered in trypanosomes n
Now known to occur in many organisms n
Refer to Table 12.5 for a list of examples
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12-77