1. PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 12
GENE TRANSCRIPTION AND RNA MODIFICATION
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2. INTRODUCTION
At the molecular level, a gene is a segment of DNA used to make a functional product
either an RNA or a polypeptide
Transcription is the first step in gene expression
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3. Gene Expression
Structural genes encode the amino acids of a polypeptide
Transcription of a structural gene produces messenger RNA, usually called mRNA
The mRNA sequence determines the amino acids in the polypeptide
The function of the protein determines traits
This path from gene to trait is called the central dogma of genetics
Refer to Figure 12.1
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4. The central dogma of genetics
Figure 12.1
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5. 12.1 OVERVIEW OF TRANSCRIPTION
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
Figure 12.2 shows common organization of a gene
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6. Signals the end of protein synthesis
common organization of a gene
Signals the end of protein synthesis
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7. Gene Expression Requires Base Sequences
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
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8. 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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9. Figure 12.3
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10. RNA Transcripts Have Different Functions
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
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11. 12-13

13. 12.2 TRANSCRIPTION IN BACTERIA
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14. 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 12.4
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15. 12-16 Figure 12.4 The conventional numbering system of promoters
Most of the promoter region is labeled with negative numbers
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
Figure The conventional numbering system of promoters
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16. Sequence elements that play a key role in transcription
The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level
Sequence elements that play a key role in transcription
Sometimes termed the Pribnow box, after its discoverer
Figure The conventional numbering system of promoters
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17. 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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18. 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
Five subunits = a2bb’
Sigma factor
One subunit = s
These subunits play distinct functional roles
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19. 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 12.6
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20. Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements
Figure 12.6
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21. A short RNA strand is made within the open complex
The binding of the RNA polymerase to the promoter forms the closed complex
Then, the open complex is formed when the TATAAT box in the -10 region 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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22. Figure 12.7
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23. 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 12.8 depicts the key points in the synthesis of the RNA transcript
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24. Similar to the synthesis of DNA via DNA polymerase
Figure 12.8
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25. 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)
2. rho-independent termination
Does not require r
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26. Rho protein is a helicase
rho utilization site
Rho protein is a helicase
r-dependent termination
Figure 12.10
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27. r-dependent termination
Figure 12.10
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28. 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
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
r-independent termination
Figure 12.11
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29. 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 and cells
Cellular complexity such as organelles
added complexity means more genes
Multicellularity
increased regulation to express only in right cells at right time
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30. 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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31. 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 12.12
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33. 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
Regulatory elements
TATA box
Transcriptional start site
Refer to Figure 12.13
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34. The core promoter is relatively short It consists of the TATA box
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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35. 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 but are often found in the –50 to –100 region
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36. 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 over a particular gene
Example: TATA box, enhancers and silencers
trans-acting elements
Regulatory proteins that bind to such DNA sequences
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37. 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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38. 12-39

39. 12-40 Figure 12.14 A closed complex CTD carboxy terminal domain (CTD)
RNA pol II can now proceed to the elongation stage
Released after the open complex is formed
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41. 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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42. 12-42

43. 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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44. 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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45. 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 12.15
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46. 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
These effects may significantly alter gene expression
Figure 12.15
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47. 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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48. 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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49. 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
Refer to Table 12.3
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50. 12-50

51. Processing
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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52. 12-52 Figure 12.16 This processing occurs in the nucleolus
Functional RNAs that are key in ribosome structure
Figure 12.16
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53. Processing 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 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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54. 12-54 Figure 12.17 Found to contain both RNA and protein subunits
Covalently modified bases
However, RNA contains the catalytic ability
Therefore, it is a ribozyme
Figure 12.17
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55. 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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56. 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
Refer to Figure 12.18
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57. 12-57 Figure 12.18 RNA displacement loop
mRNA cannot hybridize to this region
Because the intron has been spliced out from the mRNA
Figure 12.18
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58. Testing the Hypothesis
The b-globin gene from the mouse contains one or more introns
Testing the Hypothesis
Refer to Figure 12.19
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59. Figure 12.19
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60. The Data
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61. This suggests that the b-globin gene contains introns
Interpreting the Data
Hybridization caused the formation of two R loops, separated by a double-stranded DNA region
This suggests that the b-globin gene contains introns
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62. 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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63. 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 12.20
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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64. Figure 12.20
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65. 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 12.20
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66. 12-66
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67. 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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68. 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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69. The pre-mRNA splicing mechanism is shown in Figure 12.22
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
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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70. Intron loops out and exons brought closer together
Figure 12.22
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71. Intron will be degraded and the snRNPs used again
Figure 12.22
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72. 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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73. 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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74. 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 12.23, capping is a three-step process
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75. Figure 12.23
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76. Figure 12.23
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77. 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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78. 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 12.24
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79. 12-79 Figure 12.24 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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