lecture 3 gene transcription and rna modification (chapter 12)

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

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• 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

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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.

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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

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Figure 12.3

Transcription

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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!

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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

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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

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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


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

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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

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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


Initiation of Bacterial Transcription

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Figure 12.6

Binding of factor protein to DNA double helix

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Amino acids within the a helices hydrogen

bond with bases in the -35 and -10 promoter

sequences

Turn

α helicesbinding to themajor groove


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

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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


Promotor region

RNA polymerasecore enzyme

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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)

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Which is the template strand?Which is the coding strand?

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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

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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

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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

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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


• 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

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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

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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


Sequences of Eukaryotic Structural Genes

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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


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

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• 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


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


PO4

TFIIH

Figure 12.14

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A closed complex

RNA pol II can now proceed to the

elongation stageReleased after the open complex is

formed

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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)

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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

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• 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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• 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

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Focus your attention here

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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

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


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


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


Cleavage may becatalyzed by snRNAmolecules within U2and U6

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56


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


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


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


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


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–


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


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

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lecture 3 gene transcription and rna modification (chapter 12)

Documents

gene transcription

rna transcript rna polymerase

rna sequence

rna copy

messenger rna

gene polypeptide

gene expression slide

polypeptide transcription