1. RNA and Gene Expression
BIO 224
Intro to Molecular and Cell Biology

2. RNA Molecules Three major classes
Ribosomal (rRNA) make up parts of ribosomes
Messenger (mRNA) provide RNA copies of genes
Transfer (tRNA) smallest of RNAs, bring AAs to site of protein synthesis

3. Role of RNA DNA does not directly dictate protein synthesis
A molecule is needed to take information from DNA to the site of protein synthesis
RNA can be made from a DNA template
SS molecule, uses ribose as sugar, has pyrimidine U instead of T
Characteristics suggested the central dogma of the flow of genetic information in molecular biology DNARNAProtein
mRNA molecules transcribed from DNA, serve as template for translation of proteins

4. Transcription Similar to DNA replication, but different process
Makes a complementary RNA copy of the DNA strand
Entire genome never transcribed at once
Only transcribe certain genes/gene groups at certain times
RNA transcription enzymes need to recognize which gene to transcribe and where to begin
RNA polymerase enzyme transcribes RNA from DNA template: mRNA
RNA polymerase creates nucleic acid polymer of ribonucleotides
Discriminates between DNA and RNA nucleotides
Only adds ribonucleotides to polymer, creates only RNA molecule

5. 4.9 Synthesis of RNA from DNA
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6. RNA Polymerase Recognizes start and stop points of DNA molecule
Morphologically different from DNA polymerase
Both add nucleotides to 3’ OH of polymers
One recognized in E coli, several identified in eukaryotes
Made of subunits, recognizes beginning of gene
Binds over region of 60 bp or so, causes DNA to locally unwind for initiation of transcription
Promoter is region upstream or at beginning of gene that is bound
Specialized short DNA sequence
If mutated, can’t be bound, doesn’t function

7. 7.1 E. coli RNA polymerase
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8. 7.5 Structure of bacterial RNA polymerase
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9. Initiation
Promoter sequences are upstream and downstream of start site
Near promoter is closest to transcription beginning, has conserved sequence: TATAATA (TATA box)
Far promoter has conserved sequence: TTGACA
RNA subunit σ allows specific binding to promoter region sequences

10. 7.2 Sequences of E. Coli promoters
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11. Elongation
After RNA polymerase binds to promoter DNA is unwound to provide template
Unwinds near beginning of gene and provides 3’OH for template
2 RNTPs are added for transcription to begin
After addition of 10 nucleotides, σ dissociates and allows continuation of elongation

12. 7.4 Transcription by E. coli RNA polymerase (Part 1)
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13. 7.4 Transcription by E. coli RNA polymerase (Part 2)
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14. Termination
RNA polymerase recognizes termination signal to end transcription
Inverted repeat of GC rich area followed by poly-A tail transcribed to form stem-loop hairpin structure
Structure disrupts RNA association with DNA template and terminates transcription
Other method involves protein Rho that binds extended SS segments of RNA to cause termination

15. 7.6 Transcription termination
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16. Eukaryote Transcription
Similar to that in prokaryotes
Have different RNA polymerases divided into classes
Class I transcribes rRNAs
Class II transcribes mRNAs
Class III transcribes tRNAs
Mitochondria and chloroplasts have different RNA polymerases

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18. 7.11 Structure of yeast RNA polymerase II
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19. Eukaryote Initiation RNA polymerase recognizes promoter sequence
Upstream promoter sequence TATAA similar to bacterial TATA box for initiation
Also have downstream promoter element; some genes use only this for initiating transcription along with Inr
Elongation occurs in similar manner to prokaryotes

20. 7.19 A eukaryotic promoter
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21. 7.12 Formation of a polymerase II transcription initiation complex (Part 1)
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22. 7.12 Formation of a polymerase II transcription initiation complex (Part 2)
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23. 7.13 Model of the polymerase II transcription initiation complex
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24. 7.14 RNA polymerase II/Mediator complexes
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25. 7.17 Transcription of RNA polymerase III genes
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26. Messenger RNA Vary in size small to very large
RNA copy of information in DNA
Prokaryote mRNA is translated by ribosomes in the cytoplasm while still being transcribed
Prokaryotic mRNAs do not exist for long
Often degraded within minutes

27. Eukaryote mRNA
Transcribed as pre-mRNA in nucleus and processed before export
Introns removed, 5’ end capped with 7-methylguanosine, 3’ end polyadenylated with poly-A tail
Polyadenylation leads to termination of transcription, important in regulation of translation

28. 7.44 Processing of eukaryotic messenger RNAs
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29. 7.45 Formation of the 3’ ends of eukaryotic mRNAs
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30. 7.47 Splicing of pre-mRNA
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31. 7.50 Self-splicing introns
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32. 7.52 Alternative splicing in Drosophila sex determination
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33. 7.54 Editing of apolipoprotein B mRNA
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34. 7.55 Regulation of transferrin receptor mRNA stability
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35. mRNA Translation All mRNAs translated in 5’ to 3’ direction
Polypeptide chains assembled from amino to carboxy terminus
Each AA specified by an mRNA codon dictated by genetic code
Translation occurs on ribosomes, needs all three types of RNAs plus proteins

36. Expression of Genetic Information
DNA RNA Protein
Genes determine protein structure
Proteins direct cell metabolism via enzymatic activity
Genetic information specified by arrangement of DNA bases
Proteins are polymers of 20 AAs determined by sequence that dictates structure and function
Mutation in DNA sequence leads to AA sequence change: colinearity

37. 4.8 Colinearity of genes and proteins
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38. The Genetic Code
Genetic code allows understanding of how sequence of 4 nucleotides is converted to 20 AAs
tRNAs act as adaptor between AAs & mRNAs during translation
tRNA anticodons pairs in complementary fashion with mRNA codons for attachment of AA to polypeptide chain
Three nucleotides specify each AA
64 codons in genetic code: 61 code for AAs, 3 stop codons
Some AAs specified by more than one codon
Nearly all organisms use same genetic code

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40. 4.11 Genetic evidence for a triplet code
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41. 4.12 The triplet UUU encodes phenylalanine
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42. Transfer RNA
SS, folded upon themselves into DS section with cloverleaf structure
3’ end of tRNA has CCA terminus added after transcription, for AA binding
Each AA has specific tRNA
Aminoacyl tRNA synthetase enzymes attach AAs to tRNAs in two step process
tRNAs bring individual AAs to site of protein synthesis
Anticodon loop has three base anticodon sequence specific for particular complementary codon on mRNA
tRNA anticodon base pairs with mRNA codon to align AA
Redundancy of genetic code allows less stringent base pairing than in other processes
Some AAs have more than one codon, more than one tRNA

43. 4.10 Function of transfer RNA
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44. 8.1 Structure of tRNAs
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45. 7.43 Processing of transfer RNAs (Part 1)
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46. 7.43 Processing of transfer RNAs (Part 2)
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47. 8.2 Attachment of amino acids to tRNAs
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48. 8.3 Nonstandard codon-anticodon base pairing (Part 1)
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49. 8.3 Nonstandard codon-anticodon base pairing (Part 2)
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50. 8.3 Nonstandard codon-anticodon base pairing (Part 3)
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51. 8.3 Nonstandard codon-anticodon base pairing (Part 4)
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52. 8.3 Nonstandard codon-anticodon base pairing (Part 5)
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53. Protein Synthesis Similar in prokaryotes and eukaryotes
Occurs on ribosomes
Translation starts at specific sequence near 5’ end of mRNA, leaves 5’ UTR
Eukaryotic mRNAs code single polypeptide chain (monocistronic), prokaryote mRNAs code for multiple polypeptides (polycistronic)
Both prokaryote and eukaryote mRNAs have 3’ UTRs

54. 8.7 Prokaryotic and eukaryotic mRNAs
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55. Ribosomes Sites of protein synthesis in cells
Subunits separated by ultracentrifugation, mass measured in Sphedburg units: 70S in prokaryotes, 80S in eukaryotes
Made of rRNA subunits and associated proteins
Eukaryote 5S, 5.8S, 18S, 28S rRNAs all transcribed from chromosomes
Lower eukaryotes don’t produce all four
Subunits transcribed as a singe unit by RNA Pol I, to precursor that is processed into 40S and 60S subunits that make up 80S ribosome
Prokaryotes have 50S and 30S subunits of 70S ribosome
Chloroplast and mitochondrial ribosomes resemble bacterial ribosomes
Seen as a series of dots on ER, on nuclear envelope, or in cytoplasm
Cells have multiple copies of rRNA genes, actively working cells have most ribosomes

56. 7.15 The ribosomal RNA gene
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57. 7.16 Initiation of rDNA transcription
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58. 7.42 Processing of ribosomal RNAs
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59. 8.4 Ribosome structure (Part 1)
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60. 8.4 Ribosome structure (Part 2)
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61. 8.4 Ribosome structure (Part 3)
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62. 8.5 Structure of 16S rRNA
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63. 8.6 Structure of the 50S ribosomal subunit
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64. Initiation of Translation
AUG is primary start codon for translation
Translation starts with AA methionine in eukaryotes and N-formylmethionine in prokaryotes
Signals for initiation codon identification different in prokaryote and eukaryote cells
Shine-Dalgarno sequence in prokaryotes precedes mRNA initiation sequence and base pairs with sequence near 3’ terminus of 16S rRNA
Eukaryote mRNAs bound at 7-methylguanosine cap of 5’ terminus before ribosome locates initiation codon
Actual initiation of translation not entirely understood

65. 8.8 Signals for translation initiation
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66. Translation Process
Divided into stages of initiation, elongation, and termination
First step of initiation is binding of initiation factors to the small ribosomal subunit then initiator Met tRNA and mRNA
Large ribosomal subunit added to complex for elongation to proceed
Eukaryote initiation needs twelve or more proteins, eIFs , to begin
Elongation occurs after initiation, to synthesize polypeptide chain

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68. 8.9 Overview of translation
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69. 8.10 Initiation of translation in bacteria
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70. 8.11 Initiation of translation in eukaryotic cells (Part 1)
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71. 8.11 Initiation of translation in eukaryotic cells (Part 2)
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72. 8.11 Initiation of translation in eukaryotic cells (Part 3)
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73. Translation Process Three sites in ribosome for tRNA binding: P, A, E
Initiator tRNA binds to P site, second binds to A site with help of EF and peptide bond forms
Translocation moves ribosome three nucleotides along mRNA, putting empty A site over next codon, shifting peptidyl tRNA from A site to P site, and placing uncharged tRNA in E site for release
Elongation continues in this manner until stop codon moves into A site
Release factors recognize stop codons and terminate translation
mRNAs translated by series of ribosomes, sometimes simultaneously

74. 8.12 Elongation stage of translation
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75. 8.14 Termination of translation
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76. Regulation of Gene Expression
All genes not expressed at all times
All genes not expressed in all cells
Regulation of gene expression is necessary to ensure production of necessary products in correct cells at most appropriate times
Gene expression is regulated at various stages in prokaryotes and eukaryotes, with various methods being used

77. Transcriptional Regulation
Mostly occurs at level of initiation in bacteria
Negative regulation: some protein products only transcribed and translated in presence of inducers
Conserves unnecessary use of cellular energy to produce RNAs and proteins
Multiple genes expressed as operon
Operator near transcription initiation site controls transcription
Repressor binds to operator to block transcription
Inducer must be present to block repressor for transcription to occur
Operator is cis-acting, repressor is trans-acting

78. Transcriptional Regulation
Positive transcription control best characterized in E. coli by effect of glucose on genes coding for breakdown of other sugars for alternate sources of energy and carbon
If glucose available, enzymes for catabolism of other sugars not expressed
If glucose levels drop, CAP binds target sequence 60 bp upstream of transcription start site to initiate transcription of other enzymes

79. 7.7 Metabolism of lactose
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80. 7.9 Negative control of the lac operon
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81. 7.10 Positive control of the lac operon by glucose
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82. Transcriptional Regulation
More complex in eukaryotes
Controlled at initiation or elongation
Proteins bind regulatory sequences and modulate RNA polymerase activity: repressors, enhancers, promoters
Modifications of chromatin structure plays a role
Cis-acting sequences regulate eukaryote expression

83. 7.20 The SV40 enhancer
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84. 7.21 Action of enhancers (Part 1)
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85. 7.21 Action of enhancers (Part 2)
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86. 7.22 DNA looping
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87. 7.23 The immunoglobulin enhancer
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88. 7.32 Action of eukaryotic repressors
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89. 7.37 Regulation of transcription by miRNAs
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90. 7.39 DNA methylation
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91. 7.41 Maintenance of methylation patterns
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92. Translational Regulation
mRNA translation regulated in prokaryotes and eukaryotes in response to cell stress, nutrient availability, growth factor stimulation
Accomplished by repressor proteins, noncoding microRNAs, controlled polyadenylation, initiation factor modulation
mRNA localization used in eggs, embryos, nerve cells and moving fibroblasts, to allow protein production in specific locations at appropriate time

93. 8.15 Polysomes
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94. 8.16 Translational regulation of ferritin
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95. 8.17 Translational repressor binding to 3’ untranslated sequences
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96. 8.18 Localization of mRNA in Xenopus oocytes
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97. 8.19 Regulation of translation by miRNAs
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98. 8.20 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 1)
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99. 8.20 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 2)
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100. Protein Folding and Processing
Polypeptide products must be folded into 3-D conformation to function
Some protein products consist of multiple polypeptide complexes
Some proteins additionally modified by cleavage or attachment of other functional groups (carbohydrates and lipids)

101. 8.21 Action of chaperones during translation
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102. 8.22 Action of chaperones during protein transport
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103. 8.23 Sequential actions of chaperones
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104. 8.27 Proteolytic processing of insulin
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105. 8.28 Linkage of carbohydrate side chains to glycoproteins
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106. 8.34 Palmitoylation
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107. Protein Regulation
Regulation of enzyme activity necessary for proper catalysis of biological reactions
Accomplished through gene expression by regulating protein production, or regulation of protein function and activity in the cell
Allosteric binding, phosphorylation, protein degradation used to regulate enzymatic function

108. 8.36 Feedback inhibition
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109. 8.38 Protein kinases and phosphatases
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110. 8.39 Regulation of glycogen breakdown by protein phosphorylation
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111. 8.42 The ubiquitin-proteasome pathway
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112. 8.43 Cyclin degradation during the cell cycle
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113. 8.44 Autophagy
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114. Disclaimer
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