1. GENE TRANSCRIPTION AND RNA MODIFICATION
CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION

2. OVERVIEW OF TRANSCRIPTION
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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3. Signals the end of protein synthesis
• 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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4. Gene Expression Requires Base Sequences
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
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5. The 3 Stages of Transcription
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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6. 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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7. 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
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8. 12-11

9. TRANSCRIPTION IN BACTERIA 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
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10. 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 The conventional numbering system of promoters
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11. 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
The most commonly occurring bases
Figure Examples of –35 and –10 sequences within a variety of bacterial promoters
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12. Initiation of transcription - binding of RNA polymerase
Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements
Figure 12.5
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13. Then, the open complex is formed when the TATAAT box is unwound
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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14. Figure 12.6
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15. 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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16. 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
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17. Similar to the synthesis of DNA via DNA polymerase
Figure 12.7
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18. 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
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19. 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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20. Eukaryotic RNA Polymerases
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
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21. 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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22. 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
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23. The core promoter is relatively short It consists of the TATA box
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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24. 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
Silencers
Inhibit transcription
They vary in their locations but are often found in the –50 to –100 region
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25. 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
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26. 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 shows the assembly of transcription factors and RNA polymerase II at the TATA box
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27. Figure 12.12
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28. 12-37 Figure 12.12 A closed complex
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
TFIIH plays a major role in the formation of the open complex
It has several subunits that perform different functions
RNA pol II can now proceed to the elongation stage
Released after the open complex is formed
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29. Chromatin Structure and Transcription
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
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30. 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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31. Removes acetyl groups, thereby restoring a tighter interaction
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
Removes acetyl groups, thereby restoring a tighter interaction
Figure 12.13
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32. These effects may significantly alter gene expression
2. ATP-dependent chromatin remodeling
The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible
Figure 12.13
These effects may significantly alter gene expression
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33. 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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34. 12.4 RNA MODIFICATION
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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35. 12.4 RNA MODIFICATION
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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36. 12-47

37. Trimming
Many nonstructural genes are initially transcribed as a large RNA
This large RNA transcript is enzymatically cleaved into smaller functional pieces
Figure shows the processing of mammalian ribosomal RNA
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38. 12-49 Figure 12.14 This processing occurs in the nucleolus
Functional RNAs that are key in ribosome structure
Figure 12.14
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39. 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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40. 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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41. Figure 12.18
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42. 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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43. 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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44. 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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45. 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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46. 12-69 Figure 12.20 Consensus sequence in higher eukaryotes
Length varies between species
Appears to be important in the stability of mRNA and the translation of the polypeptide
From a few dozen adenines to several hundred
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47. 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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48. 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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49. The consensus sequences
Intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries
The consensus sequences
Corresponds to the boxed adenine in Figure 12.22
Sequences shown in bold are highly conserved
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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50. Intron loops out and exons brought closer together
Figure 12.22
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51. Intron will be degraded and the snRNPs used again
Figure 12.22
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52. 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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53. 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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