chapter 12 power point l

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Genetics: Analysis and PrinciplesRobert J. Brooker

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 12

GENE TRANSCRIPTION AND RNA MODIFICATION

INTRODUCTION

Transcription is the first step in gene expression

It involves two fundamental concepts 1. DNA sequences provide the underlying information

Signals for the start and end of transcription

2. Proteins recognize these sequences and carry out the process

Other proteins modify the RNA transcript to make it functionally active

12-2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Transcription literally means the act or process of making a copy

In genetics, the term refer 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


12.1 OVERVIEW OF TRANSCRIPTION

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At the molecular level, a gene is a transcriptional unit It can be transcribed into RNA

During gene expression, different types of base sequences perform different roles

Figure 12.1 shows a common organization of sequences within a bacterial gene and its transcript

Gene Expression Requires Base Sequences

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Figure 12.112-5

• Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides

• Start codon: specifies the first amino acid in a protein sequence, usually a formylmethionine (in bacteria) or a methionine (in eukaryotes)

Signals the end of protein synthesis


The strand that is actually transcribed is termed the template strand

The opposite strand is called the coding strand or the sense strand The base sequence is identical to the RNA transcript

Except for the substitution of uracil in RNA for thymine in DNA

Gene Expression Requires Base Sequences

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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

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The promoter functions as a recognition site for transcription factors

The transcription factors enable RNA polymerase to bind to the promoter forming a closed promoter complex

Following binding, the DNA is denatured into a bubble known as the open promoter complex, or simply an open complex

Initiation

Elongation

RNA polymerase slides along the DNA in an open complex to synthesize the RNA transcript

Termination

A termination signal is reached that causes RNA polymerase to dissociated from the DNA

Figure 12.2


Once they are made, RNA transcripts play different functional roles Refer to Table 12.1

A structural gene is a one that encodes a polypeptide When such genes are transcribed, the product is an RNA

transcript called messenger RNA (mRNA)

Well over 90% of all genes are structural genes

RNA Transcripts Have Different Functions

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The RNA transcripts from nonstructural genes are not translated They do have various important cellular functions

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

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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


12.2 TRANSCRIPTION 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 12.3

Promoters

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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-14

Figure 12.3 The conventional numbering system of promoters

Bases preceding this are numbered

in a negative direction

There is no base numbered 0

Bases to the right are numbered in a

positive direction

Sometimes termed the Pribnow box, after its

discoverer

Sequence elements that play a key role in transcription


Figure 12.4 Examples of –35 and –10 sequences within a variety of bacterial promoters

The most commonly occurring bases

For many bacterial genes, there is a good

correlation between the rate of RNA

transcription and the degree of agreement with the consensus

sequences


RNA polymerase is the enzyme that catalyzes the synthesis of RNA

In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme

Four subunits = 2’

Sigma factor One subunit =

These subunits play distinct functional roles

Initiation of Bacterial Transcription

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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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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-18Figure 12.5

Amino acids within the helices hydrogen

bond with bases in the promoter sequence

elements


The binding of the RNA polymerase to the promoter forms the closed complex

Then, the open complex is formed when the TATAAT box 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

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12-20Figure 12.6



The RNA transcript is synthesized during the elongation step

The DNA strand used as a template for RNA synthesis is termed the template or noncoding 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

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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 the

double helix

On average, the rate of RNA synthesis is about 43 nucleotides per second!

Figure 12.7 depicts the key points in the synthesis of the RNA transcript

Elongation in Bacterial Transcription

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Similar to the synthesis of DNA

via DNA polymerase

Figure 12.7


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

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rho utilization site

-dependent terminationFigure 12.8

Rho protein is a helicase

12-26-dependent terminationFigure 12.8

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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 Us

-independent terminationFigure 12.9

NusA

URNA-ADNA hydrogen bonds are very 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

Many of the basic features of gene transcription are very similar in bacteria and eukaryotes

However, gene transcription in eukaryotes is more complex Larger organisms Cellular complexity Multicellularity


12.3 TRANSCRIPTION IN EUKARYOTES

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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 genes And the 5S rRNA gene

Eukaryotic RNA Polymerases

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All three are very similar structurally and are composed of many subunits

There is also a remarkable similarity between the bacterial RNA pol and its eukaryotic counterparts


Eukaryotic RNA Polymerases

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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 Transcriptional start site TATA box Regulatory elements


Sequences of Eukaryotic Structural Genes

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Usually an adenine

The core promoter is relatively short It consists of the TATA box

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 12.11


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Figure 12.11

Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types

Enhancers Stimulate transcription

Silencers Inhibit transcription

They vary in their locations but are often found in the –50 to –100 region


Usually an adenine


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 on nearby

genes Example: TATA box, enhancers and silencers

trans-acting elements Regulatory proteins that bind to such DNA sequences

Sequences of Eukaryotic Structural Genes

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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

Figure 12.12 shows the assembly of transcription factors and RNA polymerase II at the TATA box

RNA Polymerase II and its Transcription Factors

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Figure 12.12


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Figure 12.12


A closed complex

TFIIH plays a major role in the formation of the open complex

It has several subunits that perform different functions

One subunit hydrolyzes ATP and phosphorylates a domain in RNA pol II known as the carboxyl terminal domain (CTD)

This releases the contact between TFIIB and RNA pol II

Other subunits act as helicases Promote the formation of the open complex

Released after the open complex is

formed

RNA pol II can now proceed to the

elongation stage


Basal transcription apparatus RNA pol II + the five GTFs

The third component for transcription is a large protein complex termed mediator It mediates interactions between RNA pol II and various

regulatory transcription factors

Its subunit composition is complex and variable

Mediator appears to regulate the ability of TFIIH to phosphorylate CTD

Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation

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The compaction of DNA to form chromatin can be an obstacle to the transcription pocess

Most transcription occurs in interphase Then, chromatin is found in 30 nm fibers that are

organized into radial loop domains Within the 30 nm fibers, the DNA is wound around histone

octamers to form nucleosomes

Chromatin Structure and Transcription

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The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs

The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol

To circumvent this problem, the chromatin structure is significantly loosened during transcription

Two common mechanisms alter chromatin structure

Chromatin Structure and Transcription

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1. Covalent modification of histones Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation

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Figure 12.13

Adds acetyl groups, thereby loosening the interaction

between histones and DNA

Removes acetyl groups, thereby restoring a tighter interaction


2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of

nucleosomes and thus make the DNA more accessible

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Figure 12.13

Proteins are members of the SWI/SNF family

Acronyms refer to the effects on yeast when these enzyme are

defectiveMutants in SWI are defective in

mating type switching

Mutants in SNF are sucrose non-fermenters

These effects may significantly alter gene expression

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 This in turn corresponds to the sequence of amino acid 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


12.4 RNA MODIFICATION

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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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Many nonstructural genes are initially transcribed as a large RNA

This large RNA transcript is enzymatically cleaved into smaller functional pieces

Figure 12.14 shows the processing of mammalian ribosomal RNA

Trimming

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Functional RNAs that are key in ribosome structure

This processing occurs in the nucleolus


Transfer RNAs are also made as large precursors These have to be cleaved at both the 5’ and 3’ ends to

produce mature, functional tRNAs

This event has been studied extensively in E. coli

Figure 12.15 shows the trimming of a precursor tRNA that carries the amino acid tyrosine (tRNAtyr) Interestingly, the cleavage occurs differently at the 5’ end

and the 3’ end

Trimming

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RNase P(Endonuclease)

Endonuclease

(RNase D)

Found to contain both RNA and protein subunits

However, RNA contains the catalytic ability

Therefore, it is a ribozyme

Covalently modified bases

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 It had been cloned earlier

Leder used a strategy that involved hybridization


Experiment 12A: Identification of Introns Via Microscopy

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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



Experiment 12A: Identification of Introns Via Microscopy

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RNA displacement loop

mRNA cannot hybridize to this region

Because the intron has been spliced out from the mRNA

The Hypothesis

The -globin gene from the mouse contains one or more introns


Testing the Hypothesis


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Figure 12.1712-56


The Data

Interpreting the Data


Hybridization caused the formation of two R loops, separated by a double-

stranded DNA region

This suggests that the -globin gene contains introns


Three different splicing mechanisms have been identified Group I intron splicing Group II intron splicing Spliceosome

All three cases involve Removal of the intron RNA Linkage of the exon RNA by a phosphodiester bond

Splicing

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Splicing among group I and II introns is termed self-splicing Splicing does not require the aid of enzymes Instead the RNA itself functions as its own ribozyme

Group I and II differ in the way that the intron is removed and the exons reconnected Refer to Figure 12.18

Group I and II self-splicing can occur in vitro without the additional proteins However, in vivo, proteins known as maturases often

enhance the rate of splicing

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Figure 12.18


Figure 12.16

In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA

Also as heterogeneous nuclear RNA (hnRNA)

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


Table 12.4 describes the occurrence of introns in genes of different species

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Most mature mRNAs have a 7-methyl guanosine 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 12.19, capping is a three-step process

Capping

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Removes one of the phosphates

Attaches GMP to the 5’ end


Attaches a methyl group to the guanine base


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

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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 12.20

Tailing

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Figure 12.20

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


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

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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

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Figure 12.21

Intron RNA is defined by particular sequences within the intron and at the intro-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


Intron loops out and exons brought closer

together

Figure 12.22


Intron will be degraded and the snRNPs used again


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?

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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?

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