CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION
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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OVERVIEW OF TRANSCRIPTION
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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
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The strand that is actually transcribed is termed the template strand (top in my diagrams)
The opposite strand is called the coding strand or the sense strand (bottom in my diagrams) 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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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
The 3 Stages of Transcription
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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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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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TRANSCRIPTION IN BACTERIA
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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
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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
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
Initiation of transcription - binding of RNA polymerase
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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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12-20Figure 12.6
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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
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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
Termination of Bacterial Transcription
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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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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
Refer to Figure 12.10
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
Refer to Figure 12.11
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
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Usually an adenine
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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
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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
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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
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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
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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
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Figure 12.13 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
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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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12.4 RNA MODIFICATION
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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
See Next Figure….
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12.4 RNA MODIFICATION
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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
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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
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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
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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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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
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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
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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 intron-exon boundaries
The consensus sequences
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
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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
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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?
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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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