Chapter 12 Power Point L

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Genetics: Analysis and Principles

Robert J. Brooker
CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
12-2
12.1 OVERVIEW OF TRANSCRIPTION
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

12-3
Gene Expression Requires Base Sequences
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
12-4
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
Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides
Figure 12.1
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Gene Expression Requires Base Sequences
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
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The Stages of Transcription
Transcription occurs in three stages

Initiation Elongation Termination
These steps involve protein-DNA interactions
Proteins such as RNA polymerase interact with DNA sequences
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Initiation
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
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
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RNA Transcripts Have Different Functions
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
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RNA Transcripts Have Different Functions
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

12-10
12-11
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
12-12
Promoters
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
12-13
Sequence elements that play a key role in transcription
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
Figure 12.3 The conventional numbering system of promoters

12-14
For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences
The most commonly occurring bases
Figure 12.4 Examples of 35 and 10 sequences within a variety of

bacterial promoters
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Initiation of Bacterial Transcription
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 = a2bb
Sigma factor
One subunit = s
These subunits play distinct functional roles

12-16
Initiation of Bacterial Transcription
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 12.5

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Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements
Figure 12.5
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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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Figure 12.6
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Elongation in Bacterial Transcription
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
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Elongation in Bacterial Transcription
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
12-22
Similar to the synthesis of DNA via DNA polymerase
Figure 12.7
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Termination of Bacterial Transcription
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) Does not require r

2. rho-independent termination
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rho utilization site
Rho protein is a helicase
Figure 12.8
r-dependent termination
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Figure 12.8
r-dependent termination
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r-independent termination is facilitated by two sequences in the RNA 1. A uracil-rich sequence located at the 3 end of the RNA 2. A stem-loop structure upstream of the Us
NusA Stabilizes the RNA pol pausing
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
Figure 12.9
r-independent termination
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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 organisms Cellular complexity Multicellularity
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Eukaryotic RNA Polymerases
Nuclear DNA is transcribed by three different RNA polymerases
RNA pol I
Transcribes all rRNA genes (except for the 5S rRNA) Transcribes all structural genes
RNA pol II
Thus, synthesizes all mRNAs
Transcribes some snRNA genes Transcribes all tRNA genes And the 5S rRNA gene
RNA pol III

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Eukaryotic RNA Polymerases
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 Refer to Figure 12.10
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Sequences of Eukaryotic Structural Genes
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 Refer to Figure 12.11
12-31
Figure 12.11
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
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Figure 12.11
Usually an adenine
Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types Enhancers
Stimulate transcription Inhibit transcription
Silencers
They vary in their locations but are often found in the 50 to 100 region
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Sequences of Eukaryotic Structural Genes
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

12-34
RNA Polymerase II and its Transcription Factors
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
12-35
Figure 12.12
12-36
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 RNA pol II can now proceed to the elongation stage
Released after the open complex is formed
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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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12-39
Chromatin Structure and Transcription
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
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Chromatin Structure and Transcription
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
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1. Covalent modification of histones
Amino terminals of histones are modified in various ways
Acetylation; phosphorylation; methylation

Adds acetyl groups, thereby loosening the interaction between histones and DNA
Figure 12.13
Removes acetyl groups, thereby restoring a tighter interaction
12-42
2. ATP-dependent chromatin remodeling
The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible
Proteins are members of the SWI/SNF family Acronyms refer to the effects on yeast when these enzyme are defective Mutants in SWI are defective in mating type switching
Mutants in SNF are sucrose non-fermenters
Figure 12.13
These effects may significantly alter gene expression

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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 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
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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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Trimming
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
12-48
This processing occurs in the nucleolus
Functional RNAs that are key in ribosome structure
Figure 12.14
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Trimming
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
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Endonuclease
(Endonuclease) RNase P
(RNase D)
Found to contain both RNA and protein subunits However, RNA contains the catalytic ability
Therefore, it is a ribozyme
Covalently modified bases
Figure 12.15
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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 b-globin gene
It had been cloned earlier
Leder used a strategy that involved hybridization
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Experiment 12A: Identification of Introns Via Microscopy
Double-stranded DNA of the cloned b-globin gene is first denatured
Then mixed with mature b-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

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RNA displacement loop
mRNA cannot hybridize to this region Because the intron has been spliced out from the mRNA
Figure 12.16
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The Hypothesis
The b-globin gene from the mouse contains one or more introns
Testing the Hypothesis
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Figure 12.17
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The Data
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Interpreting the Data
Hybridization caused the formation of two R loops, separated by a doublestranded DNA region This suggests that the b-globin gene contains introns
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Splicing
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
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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
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
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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
Figure 12.16
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Table 12.4 describes the occurrence of introns in genes of different species
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Capping
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

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Removes one of the phosphates
Attaches GMP to the 5 end
Figure 12.19
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Attaches a methyl group to the guanine base
Figure 12.19
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Capping
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
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Tailing
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

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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
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Pre-mRNA Splicing
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
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Pre-mRNA Splicing
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
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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
Figure 12.21
Serve as recognition sites for the binding of the spliceosome
The pre-mRNA splicing mechanism is shown in Figure 12.22

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Intron loops out and exons brought closer together
Figure 12.22
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Intron will be degraded and the snRNPs used again
Figure 12.22
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Intron Advantage?
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
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Intron Advantage?
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
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PowerPoint Presentation Materials

Genetics: Analysis and Principles

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

12.1 OVERVIEW OF TRANSCRIPTION

Transcription literally means the act or process of making a copy

It can continue to store information

Gene Expression Requires Base Sequences

At the molecular level, a gene is a transcriptional unit

It can be transcribed into RNA

Signals the end of protein synthesis

Gene Expression Requires Base Sequences

The strand that is actually transcribed is termed the template strand

The base sequence is identical to the RNA transcript

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

The Stages of Transcription

Transcription occurs in three stages

Initiation Elongation Termination

These steps involve protein-DNA interactions

Proteins such as RNA polymerase interact with DNA sequences

RNA Transcripts Have Different Functions

Refer to Table 12.1

A structural gene is a one that encodes a polypeptide

RNA Transcripts Have Different Functions

The RNA transcripts from nonstructural genes are not translated

For example Ribosomes Spliceosomes Signal recognition particles

12.2 TRANSCRIPTION IN BACTERIA

Promoters are DNA sequences that promote gene expression

Sequence elements that play a key role in transcription

Bases to the right are numbered in a positive direction

Sometimes termed the Pribnow box, after its discoverer

Figure 12.3 The conventional numbering system of promoters

The most commonly occurring bases

Figure 12.4 Examples of 35 and 10 sequences within a variety of

Initiation of Bacterial Transcription

Four subunits = a2bb

These subunits play distinct functional roles

Initiation of Bacterial Transcription

Refer to Figure 12.5

The sigma factor is released at this point

This marks the end of initiation

Elongation in Bacterial Transcription

It has the same base sequence as the RNA transcript

Except that T in DNA corresponds to U in RNA

Elongation in Bacterial Transcription

Similar to the synthesis of DNA via DNA polymerase

Termination of Bacterial Transcription

Termination is the end of RNA synthesis

E. coli has two different mechanisms for termination

Requires a protein known as r (rho) Does not require r

rho utilization site

Rho protein is a helicase

NusA Stabilizes the RNA pol pausing

URNA-ADNA hydrogen bonds are very weak

12.3 TRANSCRIPTION IN EUKARYOTES

Larger organisms Cellular complexity Multicellularity

Eukaryotic RNA Polymerases

Nuclear DNA is transcribed by three different RNA polymerases

Thus, synthesizes all mRNAs

RNA pol III