1. The Genetic Code and Transcription

2. 13.1 The Genetic Code Uses Ribonucleotide Bases as "Letters"
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3. The sequence of RNA is derived from the complementary bases in the DNA
Section 13.1
The genetic code is written in linear form, using the ribonucleotide bases that compose mRNA molecules as "letters"
The sequence of RNA is derived from the complementary bases in the DNA
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4. In the mRNA, triplet codons specify one amino acid
Section 13.1
In the mRNA, triplet codons specify one amino acid
The code contains "start" and "stop" signals, certain codons (nonsense codons) that are necessary to initiate and to terminate translation
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5. The triplet code provides 64 codons to specify the 20 amino acids
Section 13.2
The triplet code provides 64 codons to specify the 20 amino acids
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6. Section 13.1 The genetic code is unambiguous degenerate commaless
nonoverlapping
nearly universal
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7. Thus, the code is nonoverlapping and commaless
Section 13.2
The genetic code reads three nucleotides at a time in a continuous, linear manner
Thus, the code is nonoverlapping and commaless
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8. The nonsense codons do not specify an amino acid
Section 13.4
The genetic code is degenerate, with many amino acids specified by more than one codon
Only tryptophan and methionine are encoded by a single codon (Figure 13.7)
The nonsense codons do not specify an amino acid
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9. Figure 13-7 The coding dictionary
Figure 13-7 The coding dictionary. AUG encodes methionine, which initiates most polypeptide chains. All other amino acids except tryptophan, which is encoded only by UGG, are represented by two to six codons. The codons UAA, UAG, and UGA are termination signals and do not encode any amino acids.
Figure 13.7
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10. Section 13.4
The wobble hypothesis predicts that the initial two ribonucleotides of triplet codes are often more critical than the third. The third position of the codon-anticodon interaction would be less spatially constrained and need not adhere as strictly to the established base-pairing rules at the third position of the codon (Table 13.4)
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11. Table 13-4 Anticodon–Codon Base-Pairing Rules
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12. How do we know when to start transcription?
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13. Start (Initiation) Codon
The initial amino acid incorporated into all proteins is a modified form of methionine—N-formylmethionine (fmet)
AUG is the only codon to encode for methionine
When AUG appears internally in mRNA, an unformylated methionine is inserted into the protein
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14. How do we stop transcription?
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15. Section 13.4
Three codons (UAG, UAA, and UGA) serve as termination codons and do not code for any amino acid
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16. 13.6 The Genetic Code Is Nearly Universal
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17. Section 13.6
Mitochondrial DNA revealed some exceptions to the universal genetic code (Table 13.5)
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18. Table 13-5 Exceptions to the Universal Code
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19. What do these differences imply?
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20. Eukaryotic Transcription
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21. Transcription Synthesizes RNA on a DNA Template
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22. RNA is synthesized on a DNA template by the process of transcription
Section 13.8
RNA is synthesized on a DNA template by the process of transcription
The genetic information stored in DNA is transferred to RNA, which serves as the intermediate molecule between DNA and proteins
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23. Why can’t we just us the DNA directly?
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24. RNA polymerase directs the synthesis of RNA using a DNA template.
Section 13.10
RNA polymerase directs the synthesis of RNA using a DNA template.
No primer is required for initiation,
The enzyme uses ribonucleotides instead of deoxyribonucleotides.
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25. Section 13.11
Eukaryotes possess three forms of RNA polymerase, each of which transcribes different types of genes (Table 13.7)
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26. Table 13-7 RNA Polymerases in Eukaryotes
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27. Functional RNAs
Functional RNAs do not encode proteins, but instead perform functional roles in the cell
Transfer RNAs (tRNAs) are encoded in dozens of forms and are responsible for binding an amino acid and depositing it for inclusion into a growing protein chain 27

28. Additional Functional RNAs
Ribosomal RNA (rRNA) combines with numerous proteins to form ribosomes
Small nuclear RNA (snRNA) of various types is found in the nucleus of eukaryotes and plays a role in mRNA processing 28

29. Section 13.11
RNA polymerase II (RNP II) is responsible for a wide range of genes in eukaryotes
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30. Steps in Eukaryotic Transcription
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31. This transition is referred to as chromatin remodeling
Section 13.11
Eukaryotic transcription requires chromatin to become uncoiled, making the DNA accessible to RNA polymerase and other regulatory proteins.
This transition is referred to as chromatin remodeling
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32. Section 13.11
Eukaryotic RNA polymerases rely on transcription factors (TFs) to scan and bind to DNA
In addition to promoters, enhancers and silencers also control transcription regulation
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33. Eukaryotic mRNAs require processing to produce mature mRNAs.
Section 13.11
Eukaryotic mRNAs require processing to produce mature mRNAs.
Addition of a 5′ cap
Addition of a 3′ tail
Excision of introns
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34. Transcriptional Regulatory Interactions
Three sets of regulatory DNA sequences are commonly involved in eukaryotic gene regulation
The core promoter region is immediately adjacent to the start of transcription
These bind RNA polymerase II and associated transcription factors
Upstream of the core promoter region are various proximal elements that bind regulatory proteins
The last group of regulatory sequences are “enhancers” 34

35. 35

36. Cis-Acting Regulatory Sequences and Trans-Acting Proteins
All three regulatory regions contain cis-acting regulatory sequences, which regulate transcription of genes on the same chromosome as the sequences
The regulatory sequences bind trans-acting regulatory proteins, which can bind to their target sequences on any chromosome
At enhancers, aggregations of multiple proteins form large complexes called enhanceosomes 36

37. 37

38. The consensus sequence is 5-TATAAA-3
Promoter Elements
The most common eukaryotic promoter consensus sequence is the TATA box, or the Goldberg-Hogness box, located at about position 25
The consensus sequence is 5-TATAAA-3
A CAAT box is often found near the -80 position
A GC-rich box (consensus 5-GGGCGG-3) is located at 90, or further upstream 38

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40. Eukaryotic Promoter Elements
Eukaryotic promoters display a high degree of variability in type, number, and location of consensus sequence elements
The TATA box is most common, whereas the CAAT box and GC-rich box are more variable 40

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42. The TATA box is the principle binding site during promoter recognition
RNA pol II recognizes and binds to promoter sequences with the aid of proteins called transcription factors (TFs)
TFs bind to regulatory sequences and interact directly, or indirectly, with RNA polymerase; those interacting with pol II are called TFII factors
The TATA box is the principle binding site during promoter recognition 42

43. Promoter Recognition, continued
At the TATA box, TFIID, a multisubunit protein binds the TATA box sequence
The assembled TFIID bound to the TATA box forms the initial committed complex
Next, TFIIB, TFIIF, and RNA pol II join the complex to form the minimal initiation complex 43

44. Promoter Recognition, continued
The minimal initiation complex is joined by TFIIE and TFIIH to form the complete initiation complex
The complete initiation complex contains multiple proteins commonly referred to as “general transcription factors”
The complete complex directs RNA pol II to the 1 position, where it begins to assemble mRNA 44

45. 45

46. Enhancers and Silencers
Promoters alone may not be sufficient to initiate eukaryotic transcription
Two categories of DNA regulatory sequences lead to differential expression of genes
These are enhancer sequences and silencer sequences 46

47. Enhancer Sequences
Enhancer sequences increase the level of transcription of specific genes
They bind proteins that interact with the proteins that are bound to gene promoters, and together the promoters and enhancers drive gene expression
Enhancers may be variable distances from the genes they affect and may be upstream or downstream of the gene 47

48. Enhancer Sequences and DNA Bending
Enhancer sequences bind activator proteins and associated coactivators that form a “protein bridge” that links the proteins at the enhancer sequence to the initiation complex at the promoter
This bridge bends the DNA so that the proteins at both locations are brought close enough together for them to interact
This bridge is known as an “enhanceosome” 48

49. 49

50. These bends reduce transcription of the target gene
Silencer Sequences
Silencer sequences are DNA elements that act at a distance to repress transcription of their target genes
Silencers bind transcription factors called repressor proteins that induce bends in DNA
These bends reduce transcription of the target gene
Silencers may be located variable distances from their target genes, either upstream or downstream 50

51. Insulator Sequences
Insulator sequences are cis-acting sequences located between enhancers and the promoters of genes that need to be protected from the action of the enhancers
Insulators ensure that only the target gene is regulated by the enhancer
Insulators may allow formation of DNA loops that contain the enhancers and their intended target promoters while excluding nontarget genes 51

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53. ANIMATION: mRNA Production in Eukaryotes53

54. Post-Transcriptional Processing
The initial eukaryotic gene mRNA is called the pre-mRNA whereas the fully processed mRNA is called the mature mRNA; modifications include
5 capping
3 polyadenylation
Intron splicing 54

55. Capping 5 mRNA
After the first 20 to 30 nucleotides of mRNA have been synthesized, a special enzyme, guanylyl transferase, adds a guanine to the 5 end of the pre-mRNA
Additional enzyme action methylates the newly added guanine and may also methylate nearby nucleotides of the transcript 55

56. Protection of mRNA from rapid degradation
Functions of the 5 Cap
Protection of mRNA from rapid degradation
Facilitating transport of mRNA out of the nucleus
Facilitating subsequent intron splicing
Enhancing translation efficiency by orienting the ribosome on the mRNA 56

57. Polyadenylation of 3 Pre-mRNA
Termination of transcription by RNA pol II is not fully understood
The 3 end of the pre-mRNA is created by enzyme action that removes a section of the 3 message and replaces it with a string of adenines
This is thought to be associated with the subsequent termination of transcription 57

58. 58

59. Functions of Polyadenylation
Facilitating transport of mature mRNA across the nuclear membrane to the cytoplasm
Protecting the mRNA from degradation
Enhancing translation by enabling the ribosomal recognition of mRNA
Some eukaryotic transcripts (e.g., the histone genes) do not undergo polyadenylation 59

60. Section 13.12
Introns (intervening sequences) are regions of the initial RNA transcript that are not expressed in the amino acid sequence of the protein
Roberts and Sharp shared the 1993 Nobel Prize for their codiscovery of “split genes,” i.e., the presence of intron and exon sequences
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61. Section 13.12
Introns are removed by splicing, and the exons are joined together in the mature mRNA
The size of the mature mRNA is usually much smaller than that of the initial RNA (Figure and Table 13.8)
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62. Figure 13.12
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63. Table 13-8 Contrasting Human Gene Size, mRNA Size, and the Number of Introns
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64. Pre-mRNA Intron Splicing
Intron splicing requires great precision to remove intron nucleotides accurately
Errors in intron removal would lead to incorrect protein sequences 64

65. Splicing Signal Sequences
Specific short sequences define the junctions between introns and exons
The 5 splice site
The 3 splice site
The Branch Site 65

66. 66

67. Introns are removed by an snRNA-protein complex called the spliceosome
Splicing
Introns are removed by an snRNA-protein complex called the spliceosome
Like molecular “workbench”
The 5 splice site is cleaved first
Then the 3 splice site is cleaved and the exon ends are ligated together 67

68. ANIMATION: RNA Splicing68

69. May play a roll in alternative intron splicing
Accurate Splicing
Spliceosome components are recruited to 5 and 3 splice sites by SR proteins (pathfinders)
May play a roll in alternative intron splicing 69

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71. Gene Expression Machines
Current models suggest that RNA pol II and an array of pre-mRNA processing proteins function as “gene expression machines”
The proteins that carry out capping, intron splicing, and polyadenylation associate with the CTD of pol II
All three processes are carried out simultaneously 71

72. 72

73. ANIMATION: RNA Processing Control73

74. The Human Genome
Humans produce more than 100,000 distinct peptides
Assumed 80, ,000 genes
We were wrong
~22,000 genes in humans
What’s going on?

75. Alternative Transcripts of Single Genes
It is common for large eukaryotic genomes to express more proteins than there are genes in the genome
Three transcription-associated mechanisms can explain this 75

76. Alternative pre-mRNA Processing
pre-mRNA can be spliced in alternative patterns in different cell types
Alternative promoters can initiate transcription at distinct start points
Different start point
Alternative localizations of polyadenylation can produce different mature mRNAs
Different stop point 76

77. Alternative Intron Splicing
Alternative intron splicing: processing of identical transcripts in different cells can lead to mature mRNAs with different combinations of exons and thus different polypeptides
Approximately 70% of human genes are thought to undergo alternative splicing
It is less common in other animals and rare in plants 77

78. 78

79. Not all of the possible arrangements are observed, however
Dscam
The Drosophila Dscam gene has one of the most complex patterns of alternative splicing
Of the 24 exons, numbers 4, 6, 9, and 17 have numerous alternative sequences
More than 38,000 different polypeptides can be produced through alternative splicing
Not all of the possible arrangements are observed, however 79

80. 80

81. Alternative Processing
Alternative splicing is mainly controlled by variation in SR proteins in different cell types
Use of alternative promoters
More than one sequence upstream of a gene can initiate transcription
Alternative polyadenylation
Alternative promoters of polyadenylation 81

82. Five polyadenylation sites Yields 9 mature mRNA
Rat α-tropomyosin
14 exons
With 4 alternates
Two promoters
Five polyadenylation sites
Yields 9 mature mRNA

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