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Genetics: Analysis and Principles
Robert J. Brooker
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION (processing)
Figure 12.112-5
The central dogma of genetics
A key concept is that DNA base sequences define the beginning and end of a gene and regulate the level of RNA synthesis
Gene expression is the overall process by which the information within a gene is used to produce a functional product which can determine a trait in play with the environment
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
12.1 OVERVIEW OF TRANSCRIPTION
12-6
Figure 12.212-7
Signals the end of protein synthesis
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The strand that is actually transcribed (used as the template) 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
Transcription factors recognize the promoter and regulatory sequences to control transcription
mRNA sequences such as the ribosomal-binding site and codons direct translation
Gene Expression Requires Base Sequences
12-8
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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 Transcription factors that control transcription bind
directly or indirectly to DNA
The Stages of Transcription
12-9
12-10Figure 12.3
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Once they are made, RNA transcripts play different functional roles
Well over 90% of all genes are structural genes producing mRNA
The other RNA molecules are never translated: This collection appears much greater that initially believed; Some RNAs are 20-25 nts long that have important functions!
RNA Transcripts Have Different Functions
12-11
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
12-12
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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12.2 TRANSCRIPTION IN BACTERIA
12-14
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Promoters are DNA sequences that “promote” gene expression: Events at this piece of DNA are needed to initiate RNA synthesis/transcription More precisely, they direct the exact location for the
initiation of transcription and determine when and how frequently a gene is transcribed.
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
Promoters
12-15
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-16
Figure 12.4 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
Most of the promoter region is labeled with negative numbers
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-17
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-18
Figure 12.5 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
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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 = 2’ Sigma factor
One subunit =
These subunits play distinct functional roles
Initiation of Bacterial Transcription
12-19
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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
Refer to Figure 12.6
Initiation of Bacterial Transcription
12-20
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-21Figure 12.6
Amino acids within the helices hydrogen
bond with bases in the promoter sequence
elements
12-23Figure 12.7
12-26
Similar to the synthesis of DNA
via DNA polymerase
Figure 12.8
On average, the rate of RNA synthesis is about 43 nucleotides per second!
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
12-27
12-28
rho utilization site
-dependent terminationFigure 12.10
Rho protein is a helicase
12-29-dependent terminationFigure 12.10
12-30
-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 terminationTermination in Eukaryotes is much less well defined !
Figure 12.11
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 and cells Cellular complexity such as organelles
added complexity means more genes Multicellularity: many different cell types
increased regulation to express only in right cells at right time
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12.3 TRANSCRIPTION IN EUKARYOTES
12-31
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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
12-32
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Eukaryotic promoter sequences are more variable and often much more complex than those of bacteria
For structural genes, at least three features are found in most promoters Regulatory elements TATA box (present in ~20 % of our genes) and other
short sequences in TATA-promoters that have a similar function
Transcriptional start site
Refer to Figure 12.13
Sequences of Eukaryotic Structural Genes
12-34
12-35
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 12.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
12-36
Figure 12.13
Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types
Enhancers Stimulate transcription
Silencers Inhibit transcription
They vary widely in their locations, from –50 to –100 region
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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
12-37
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
RNA Polymerase II and its Transcription Factors
12-38
12-39
Figure 12.14
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
12-40
Figure 12.14
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A closed complex
Released after the open complex is
formed
RNA pol II can now proceed to the
elongation stage
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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
12-41
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
The compaction of DNA to form chromatin can be an obstacle to the transcription process
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
12-43
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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
12-44
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1. Covalent modification of histones Amino terminals of histones are modified in various ways
Acetylation; phosphorylation; methylation
12-45
Figure 12.15
Adds acetyl groups, thereby loosening the interaction
between histones and DNA
Removes acetyl groups, thereby restoring a tighter interaction
These effects may significantly alter gene expression
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2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
12-46
Figure 12.15
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
‘promoter’ Protein coding
Difference in gene structure between
- prokaryote
- eukaryotecore
‘promoter’
An important difference between prokaryotes and eukaryotes is that eukaryotes’ genes are not split into intons and exons in eukaryotes is the DNA coding protein are. Therefore, exons eventually end up in the mRNA
intron
exons
Pre-mRNA
Transcription start, elongation, termination and RNA processing in eukaryotes
: coding protein: non-coding protein: ‘leader’ and ‘trailer’
CAP
CAP (poly A tail)
The longest gene in human genome is more than 1.500.000 base pares (bp) and the mRNA is ~ 7000 nt. That means: >1.493.000 bp intron = ~ 99,5 % !!!!!
‘promoter’
intron
exons
GENE
mRNA AAAAAAAAAAAAAAn
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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12.4 RNA MODIFICATION
12-48
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
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12.4 RNA MODIFICATION
12-49
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-65
Figure 12.20
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
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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
12-67
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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
12-68
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-69
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
12-70
Intron loops out and exons brought closer
together
Figure 12.22Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-71Figure 12.22
Intron will be degraded and the snRNPs used again
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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?
12-72
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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?
12-73
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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
Capping: marking 5’ends of mRNAs
12-74
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-75Figure 12.23
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-76Figure 12.23
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
Function of Capping
12-77
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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 3’ end of a mRNA: Tailing
12-78
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-79
Figure 12.24
Consensus sequence in higher eukaryotes
Appears to be important in the transport and stability of mRNA
and the translation of the polypeptide
Length varies between species
From a few dozen adenines to several hundred