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Transcription and translation


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Transcription and translation

  1. 1. Prokaryotic Transcription
  2. 2. Transcription <ul><li>DNA-dependent RNA synthesis </li></ul><ul><li>RNA polymerase doesn’t require a primer </li></ul><ul><li>Ribonucleotides not deoxyribonucleotides incorporated into the polymer </li></ul><ul><li>Uracil substituted for thymine </li></ul><ul><li>Template for transcription is the antisense strand </li></ul>
  3. 3. Stages <ul><li>Initiation : Occurs after promoter recognition and polymerase binding when the first rNTP is inserted </li></ul><ul><li>Elongation : adding rNTPs to chain </li></ul><ul><ul><li>Sigma subunit dissociates after a few bases are added to the chain </li></ul></ul><ul><li>Termination : dissociation of core polymerase and release of RNA transcript </li></ul>
  4. 4. Phases of transcription
  5. 5. RNA Polymerase
  6. 6. RNA Polymerase
  7. 7. E. coli RNA Polymerase <ul><li>Sigma subunit recognizes the transcription start site </li></ul><ul><li>Several different sigma subunits that recognize different promoters </li></ul>
  8. 8. Promoters <ul><li>Sequences that regulate the efficiency of transcription initiation </li></ul><ul><li>Can be strong or weak </li></ul><ul><li>Contain palindromic (e.g. RADAR) consensus sequences recognized by sigma subunit </li></ul><ul><li>TTGACA – sigma 70 subunit recognition domain is always at -35 </li></ul><ul><li>TATAAT - Pribnow (or TATA) box  always at -10 for unwinding of helix </li></ul><ul><li>Distance between TATAAT and TTGACA very important for polymerase binding (~17 bp) </li></ul>
  9. 9. Prokaryotic Promoters <ul><li>-35 and 10 regions recognized by regions 2 & 4 of σ 70 factor </li></ul><ul><li>extended -10 recognized by region 3 </li></ul><ul><li>Region 4 has helix-turn-helix DNA binding motif </li></ul><ul><ul><li>1 helix interacts with major groove at -35 & other lies on top of groove interacting with bases </li></ul></ul><ul><ul><li> helix interacts with -10 region on nontemplate strand </li></ul></ul><ul><ul><li>UP-element recognized by  CTD </li></ul></ul>=
  10. 10. Sigma 70 binding sites Region 3.2 acts as molecular mimic in abortive initiation; region 2.3 melts DNA
  11. 11. Open complex- note regions of binding by σ 70 binds to UP-element
  12. 12. The Transcription Unit
  13. 13. Transcription Occurs in a Bubble
  14. 14. Synthesis Occurs in the 5 ´ to 3´ Direction
  15. 15. Initiation
  16. 17. Transcription Requires Gyrase and Topoisomerase
  17. 18. Termination <ul><li>Intrinsic termination - caused by a palindromic sequence in the DNA template that results in the formation of a hairpin loop that prevents elongation </li></ul><ul><li>Rho-dependent termination -  protein physically interacts with RNA transcript preventing elongation. Most gene transcription is terminated this way in prokaryotes </li></ul><ul><li>Antitermination - viral protein allows polymerase to read through the termination sequence making a different protein </li></ul>
  18. 19. Intrinsic Termination
  19. 20. Rho-Dependent Termination
  20. 21. Antitermination
  21. 22. Eukaryotic Transcription
  22. 23. Regulation of Eukaryotic Gene Expression
  23. 24. Transcription Results in an Unprocessed Message 5 ´ Cap added immediately to 5 ´ sequence, in this case ACATTTG Poly(A) tail added when sequence (5  AATAAA 3  ) is transcribed Heterogeneous nuclear RNA (hnRNA) also known as (aka) pre-mRNA or the primary transcript
  24. 25. Translation of mRNA yields Protein
  25. 26. Eukaryotes Have 3 RNA Polymerases <ul><li>Pol I synthesizes rRNA in the nucleolus, not inhibited by  -amanitin (octapeptide synthesized by a mushroom) </li></ul><ul><li>Pol II synthesizes mRNA and snRNA, inhibited by low concentrations of  -amanitin </li></ul><ul><li>Pol III synthesizes 5s rRNA and tRNA, inhibited by high concentrations of  -amanitin </li></ul>
  26. 27. RNA Polymerase II <ul><li>12 subunits shaped like a crab claw </li></ul><ul><li>Jaws grip template & clamp locks template at catalytic site for high processivity </li></ul><ul><li>1 Mg 2+ at catalytic site </li></ul><ul><li>8-9 bp of hybrid puts 3 ´ OH at catalytic site </li></ul><ul><li>20 bp DNA downstream in cleft </li></ul><ul><li>RNA fits in grooves </li></ul>
  27. 28. <ul><li>Several channels lead to the active site </li></ul><ul><li>2 DNA channels up and downstream from transcription bubble make DNA bend about 90° </li></ul><ul><li>Tunnel on opposite side of DNA entry for NTP diffusion and incorporation into RNA </li></ul><ul><li>Rudder protrudes from active site to split RNA-DNA hybrid </li></ul><ul><li>RNA exits from another channel opposite </li></ul><ul><li>DNA entry that has a protein flap that may aid in elongation and termination </li></ul>
  28. 29. A Typical Gene Transcribed by RNAP II
  29. 30. Anatomy of a Gene
  30. 31. Transcription Initiation Promoters
  31. 32. Overview <ul><li>Elements in the promoter can be common to all genes and used constitutively </li></ul><ul><li>Other elements are gene specific: identify particular classes of genes </li></ul><ul><li>These elements exist in different combinations in individual genes </li></ul><ul><li>Housekeeping genes contain elements recognized by general and upstream factors and are transcribed in all cells </li></ul>
  32. 33. Cis-acting Elements Located at a Fixed Distance from Initiation Site <ul><li>General elements bound by basal factors for initiation are called Consensus Sequences </li></ul><ul><li>GC box: -110 = GGGCGG, often multiple copies </li></ul><ul><li>CAAT box: -80 = GGCCAATCT </li></ul><ul><li>TATA Box: -30 (Goldberg-Hogness Box) = TATAAAA is pretty nonspecific, fixes initiation site because it is easily denatured </li></ul><ul><li>RNAP II binds at TATA Box </li></ul>
  33. 34. The Promoter Binds General Transcription Factors & RNAP II
  34. 35. In vitro Mutagenesis Shows Critical Sequences for Transcription Initiation
  35. 36. Enhancers <ul><li>DNA sequences that can modulate transcription from a distance </li></ul><ul><li>Can be upstream, close to start, or down stream of start site </li></ul><ul><li>Aren't always directly involved in template binding, but are essential to efficient transcription </li></ul><ul><li>Can be negative or positive, but are usually positive </li></ul><ul><li>Position is not fixed - can be upstream, downstream or within an intron </li></ul>
  36. 37. <ul><li>Can be removed and put back into a different gene and work </li></ul><ul><li>Can be inverted with no effect on activity </li></ul><ul><li>Control chromatin structure and rate of transcription (affect efficiency and stimulate) </li></ul><ul><li>Not necessary for transcription but are necessary for full activation (basal vs. induced expression) </li></ul><ul><li>Are responsible for time and tissue-specific gene expression </li></ul><ul><li>Interact with regulatory proteins & transcription factors </li></ul>
  37. 38. Enhancer Sequences (Response Elements)
  38. 39. Upstream Elements Vary with Gene Function
  39. 40. Modular Nature of Upstream Region for Tissue-Specific Gene Expression Note that many different transcription factors can bind to one gene It is the set of proteins bound to a gene that determine the level and location of gene expression
  40. 41. Bending DNA Stimulates Transcription
  41. 42. Transcription Factors <ul><li>Trans-acting factors </li></ul><ul><li>Directly facilitate template binding </li></ul><ul><li>Essential for transcription initiation because RNAP II can't bind to promoter and start transcription because eukaryotic chromatin is complexed to protein and promoters are hidden. </li></ul><ul><li>The &quot;  factor&quot; for eukaryotes. </li></ul><ul><li>Different transcription factors may compete for promoter/ binding elements, some of which overlap. </li></ul><ul><li>Concentration and affinity effect which binds. </li></ul><ul><li>Sometimes same binding element binds different transcriptional factors in a tissue specific manner. </li></ul><ul><li>Some are gene specific. </li></ul>
  42. 43. <ul><li>Basal Factors = TFII s - always associated with Pol II </li></ul><ul><li>True Activators : modular proteins with 2 domains </li></ul><ul><li>DNA-Binding Domain : DNA-Protein Interaction </li></ul><ul><ul><li>Distinct structural motifs </li></ul></ul><ul><ul><li>DNA-Binding Domains are classified by structural motif </li></ul></ul><ul><li>Trans-activating Domain : Protein-Protein Interaction </li></ul><ul><li>Can interact with RNAP II or other transcription factors at the promoter and coactivators (hormones, small metabolites) </li></ul>
  43. 44. General (Basal) TFs
  44. 45. Pol II Core Promoter <ul><li>Core promoter – minimal set of sequences necessary for accurate initiation </li></ul><ul><li>BRE – TFIIB recognition element </li></ul><ul><li>TATA Box </li></ul><ul><li>Inr- Initiator </li></ul><ul><li>DPE – Downstream element </li></ul><ul><li>Typical promoter usually include only 2 or 3 of any of these </li></ul><ul><li>Upstream lie regulatory elements </li></ul>
  45. 46. Stages of Initiation
  46. 47. Commitment <ul><li>TFII D complex binds to TATA box via TATA Box Binding Protein (TBP) and TAF's (TATA Associated Factors) ~20 bases involved </li></ul><ul><li>TFII D complex contacts DNA - changes conformation to facilitate binding of TFII B and A </li></ul><ul><li>RNAP II complexed to TFII F binds next </li></ul><ul><li>TFII E </li></ul><ul><li>TFII H (helicase & kinase activity) </li></ul><ul><li>NTPs enter </li></ul><ul><li>TFII J (?) </li></ul>
  47. 48. Assembling the Basal Complex
  48. 49. Initiation
  49. 50. TAFs Interact with TFIID (& TBP)
  51. 52. Mediator Complexes <ul><li>Multiprotein complexes associated with RNAP II </li></ul><ul><li>Do not bind to DNA </li></ul><ul><li>Act like control panels for RNAP II </li></ul><ul><li>Mediate interactions with TFs </li></ul><ul><li>Often required for the function of TFs </li></ul><ul><li>Integrate all of the positive and negative regulatory signals for RNAP II and &quot;determine&quot; how much message should be made. </li></ul><ul><li>Probably interact with the C-terminal domain (CTD) of the largest RNAP II subunit </li></ul>
  52. 53. Mediator Complexes Are Composed of Many Coactivators
  53. 54. Coactivators Interact with TFs, But Do Not Bind to DNA
  54. 55. Mediators May Stabilize Pre-Initiation Complex After Chromatin Remodeling <ul><li>Mediators associate with the CTD tail </li></ul><ul><li>Interact with DNA-bound activators </li></ul>
  55. 57. Multiple Pathways Affect Transcriptional Activation
  56. 58. Promoter Escape <ul><li>Pol II moves away from the promoter </li></ul><ul><li>Synthesizes 10-15 nucleotides </li></ul><ul><li>Dissociates from general initiation factors </li></ul><ul><li>Cannot occur unless CTD is hyperphosphorylated by TFII H </li></ul>
  57. 59. Promoter Escape Requires CTD Phosphorylation by TFIIH
  58. 60. Other Proteins Associated with Promoter Escape & Elongation
  59. 61. Elongation Phosphorylation of Ser 2 recruits splicing factors Phosphorylation of Ser 5 recruits capping factors Other factors include TFIIS, P-TEFb, TAT-SF1
  60. 62. Abortive Transcriptionand Proofreading <ul><li>Transcripts smaller than about 9 nucleotides are aborted </li></ul><ul><li>TFII F acts to decrease abortive transcription (by increasing rate of polymerization?) </li></ul><ul><li>TFIIS contributes to Pol II’s proofreading by stimulating its inherent RNase activity </li></ul>
  61. 63. Arrested Transcription <ul><li>Transcription can also be arrested at promoter escape, potentially by TFII F binding to promoter ahead of transcriptional start site </li></ul><ul><li>Can be suppressed by TFII E & TFII H-XPB DNA helicase (ATP-dependent) activity which act to disrupt TFII F's interaction with the promoter </li></ul><ul><li>TFII H also recognizes damaged template DNA and recruits proteins for DNA excision-repair </li></ul><ul><li>Mutations in TFII H can result in diseases with sensitivity to light and increased risk of cancer such as xeroderma pigmentosum, trichthiodystrophy, or Cockayne syndrome (depending on mutation severity) </li></ul>
  62. 64. EFs Can Reactivate Arrested RNAP II <ul><li>The SII family of EFs reactivate stalled Pol II by cleaving the transcript upstream of the 3´-OH of the last nucleotide making a new 3´ end so that RNAP II can add new nucleotides </li></ul>
  63. 65. EFs Can Prevent RNAP II Arrest <ul><li>P-TEFb is a cyclin-dependent kinase that phosphorylates CTD to prevent elongation arrest </li></ul><ul><li>DSIF and NELF are negative regulators of elongation </li></ul><ul><ul><li>Both interact with Pol II in its hypophosphorylated form </li></ul></ul><ul><ul><li>DSIF/NELF blockade is removed by P-TEFb phosphorylation of CTD </li></ul></ul>
  64. 66. RNAP II Pausing Is the Rate-limiting Step in Elongation <ul><li>EFs can prevent pausing </li></ul><ul><li>TFII F, ELL, Elongin & CSB suppress pausing by decreasing the time RNAP II spends in an inactive conformation increasing rate the of transcription </li></ul>
  65. 67. EFs Modify Chromatin Structure <ul><li>HMG14, FACT & Elongator modify and destabilize nucleosomes clearing the path for RNAP II movement </li></ul><ul><li>Elongator and SWI/SNF remodel chromatin </li></ul>
  66. 68. Elongation
  67. 69. Transcription Visualized Prokaryotic Eukaryotic
  68. 70. RNA processing is coupled to elongation <ul><li>RNAP II CTD interacts with RNA processing proteins to process the transcript as it comes through the flap at the end of tunnel </li></ul><ul><li>7-methyl guanine cap, splicing and polyadenylation are coupled to elongation </li></ul>
  69. 71. Capping the 5 ´ end of the transcript <ul><li>The 7-methyl guanosine cap is added to the 5´-PO 4 before the transcript is 30 nucleotides long </li></ul><ul><li>May be used to attenuate mRNA output </li></ul><ul><li>Unique 5  -5  bond is added shortly after transcription initiation via 3 reactions </li></ul>
  70. 72. Capping reactions <ul><li>Phosphatase removes 5  phosphate </li></ul><ul><li>Guanylyl transferase catalyzes a condensation between the 5  triphosphate and GTP </li></ul><ul><li>Guanine-7-methyltransferase transfers the methyl group </li></ul><ul><li>Ser 5 of CTD dephosphorylated and capping machinery leaves </li></ul><ul><li>All eukaryotes possess a methyl on N7 of the terminal guanine </li></ul><ul><li>Higher eukaryotes often add a second methyl group to the penultimate base at the 2  -O position (2  -O-methyltransferase) </li></ul>
  71. 73. 3  Polyadenylation <ul><li>Ser 2 must be phosphorylated for poly(A) factor recruitment </li></ul><ul><li>Length of poly(A) tail determined by proteins bound to poly(A) sequence </li></ul><ul><li>AAUAA signals the addition of the poly(A) tail </li></ul><ul><li>Poly(A) polymerase adds ~200 A residues to the free 3  -OH of the transcript </li></ul><ul><li>Poly(A) tail leads to cleavage ~10-35 upstream of signal </li></ul><ul><li>Cleavage polyadenylation stimulatory factor (CPSF) recognizes the polyadenylation sequence (AAUAAA) </li></ul><ul><ul><li>associates with TFIID first, then jumps on to CTD after initiation </li></ul></ul><ul><li>Cleavage stimulatory factor (CstF) also interacts with CTD </li></ul><ul><ul><li>necessary for elongation </li></ul></ul>
  72. 74. Poly(A) Tail Confers Stability to mRNA <ul><li>Poly(A) tail is associated with the poly(A)-binding protein (PABP) </li></ul><ul><li>Poly(A) tail + PABP thought to confer stability to many mRNA transcripts and is involved in translation initiation </li></ul>
  73. 75. RNA Splicing <ul><li>As pointed out earlier, the concept of a gene having protein-coding sequences interrupted by non-coding sequences was not recognized until the late 1970’s </li></ul><ul><li>Work in Phil Sharp’s lab at MIT by his post-doc Sarah Flint demonstrated that eukaryotic gene structure differed from prokaryotic gene structure </li></ul><ul><li>Walter Gilbert named these gene regions: </li></ul><ul><ul><li>Exon = expressed sequences </li></ul></ul><ul><ul><li>Intron = intervening sequences </li></ul></ul>
  74. 76. Splicing Visualized Transcription initiated here Introns loop out as they are excised
  75. 77. Splicing Mechanisms Introns Are Classified by Their Splicing Mechanism
  76. 78. Splicing involves two transesterifications
  77. 79. Trans-Splicing joins exons from two different RNAs
  78. 80. Self-excising group I & 2 introns 2 nucleophilic transesterification reactions 3  -OH guanosine on right side of intron is transferred to nucleotide at 5  end of intron Guanosine acts as cofactor &quot;New&quot; 3  -OH on left side of intron and phosphate group on 3  end of right side of intron interact leaving phosphate for ligation of exons 1 and 2
  79. 81. Spliceosome <ul><li>snRNA s mall n uclear RNAs </li></ul><ul><li>snRNPs small ribonucleoproteins (snurps) </li></ul><ul><ul><li>rich in uridine </li></ul></ul><ul><ul><li>only in the nucleus </li></ul></ul><ul><ul><li>designated U1, U2, etc. </li></ul></ul><ul><li>Serine-Arginine (SR) proteins act as bridging factors </li></ul><ul><ul><li>N- terminal RNA recognition motifs for binding hnRNA </li></ul></ul><ul><ul><li>C-terminal arg-ser (RS) rich sequences for protein-protein interactions with RS domains in snurps </li></ul></ul>
  80. 82. hnRNPs Involved in Splicing Reactions
  81. 83. Nuclear Splicing 1. U1 binds to exon 1-intron (5  splice site) binding site 2. U2, U4, U5 & U6 bind, splicing begins (2 trans-esterification reactions) 3. 2  -OH from branchpoint (internal adenine residue) of intron attacks 5  splice site & cuts polymer 4. Free OH created at the end of exon 1 attacks the intron-exon 2 junction 5. Introns excised 6. Exons ligated
  82. 84. Assembling the Splicesome
  83. 85. Splicing and errors <ul><li>Errors are decreased by coupling transcription and splicing – see 3 ′ site as transcribed so no competition from other sites </li></ul><ul><li>Errors decreased by exonic splicing enhancers – ser arg rich sites that are bound by the essential SR proteins that recruit snurps to splice sites </li></ul><ul><li>SR proteins also necessary for alternative splicing </li></ul>
  84. 86. Putting It All Together
  85. 87. Alternative Splicing Regulates Gene Expression
  86. 88. Alternative splicing
  87. 89. Alternative splicing results in families of proteins (splicing isoforms)
  88. 90. Types of Alternative Splicing (a) Alternative 5 ´ splice site (b) Alternative 3 ´ splice site (c) Skipping the variable alternative splice exon (d) Mutual exclusion of exons (e) Gender-specific splicing Alternative Poly(A) site Found in prostate cancer
  89. 91. Preprotachykinin (PPT) Gene P P
  90. 92. Splicing is regulated
  91. 93. Combinatorial Control Sex lethal binding results in stop codon being spliced out Functional transformer binding causes doublesex to be spliced in a female-specific fashion Stop codon remains Transformer not functional Male-specific doublesex
  92. 94. Exon shuffling
  93. 95. RNA Editing Changes the sequence of the RNA after transcription, but before translation
  94. 96. Insertion/Deletion Editing <ul><li>Nucleotide addtion or subtraction directed by guide RNA (gRNA) templates </li></ul><ul><li>Add poly(U) to form initiation codon and set reading frame </li></ul><ul><li>gRNA template complementary to edited region of final RNA transcript </li></ul><ul><li>gRNA base pairs with pre-RNA and directs editing complex to make appropriate changes to RNA transcript </li></ul>
  95. 97. gRNA Directs T. brucei RNA Editing
  96. 98. Substitution editing <ul><li>Nucleotides are altered by substituting one for another </li></ul><ul><li>Prevalent in mitochondria and chloroplasts </li></ul><ul><li>Apolipoprotein B (apo B) exists in long and short forms </li></ul><ul><li>Intestine: protein complex binds to &quot;mooring&quot; sequence downstream of editing site </li></ul><ul><li>C to U substitution: CAA = glutamine; UAA = stop (short form) </li></ul>
  97. 99. Apo-B Gene Is Modified by RNA Editing
  98. 100. Transcription-Induced Z-DNA, dsRNA & RNA Editing <ul><li>Z-DNA stabilized by negative supercoiling induced by RNAP II </li></ul><ul><li>dsRNA editing substrate forms by 3 ´ intron folding back on exon to be edited </li></ul><ul><li>A denosine D eaminase A cting on R NA (ADAR) 1 Binds to dsRNA and Z-DNA </li></ul><ul><li>It is proposed that binding to Z-DNA allosterically activates ADAR1 </li></ul><ul><li>ADAR1 deaminates adenosine to inosine </li></ul><ul><li>I read as G during translation resulting in glutamine (CAG) to arginine (CGG) substitution </li></ul><ul><li>Occurs in Glutamine Receptor-B and Serotonin-2C receptor </li></ul>
  99. 101. ADAR1 Mechanism
  100. 102. Antisense (RNAi, siRNA and miRNA) Regulation of Translation <ul><li>All use a large dsRNA that activates an enzyme called dicer </li></ul><ul><li>Dicer digests large transcript into short pieces (21-23 nt) that recruit the RISC complex of proteins </li></ul><ul><li>The RNAi (siRNA or miRNA) then bind to the target resulting in translational arrest, digestion of newly-formed dsRNA or promoter silencing via chromatin modification </li></ul>
  101. 103. RNAi/miRNA Mechanism Kosik Nature Reviews Neuroscience 7 , 911 – 920 (December 2006) | doi:10.1038/nrn2037
  102. 104. C. elegans makes lin-4 antisense to regulate lin-14 expression Now consider lin-4 antisense an miRNA
  103. 105. Have Many Antisense &quot;Drugs&quot; in Clinical Trials <ul><li>Vitravene  is an antisense “ drug ” that targets cytomeglavirus in AIDS patients with cytomeglavirus-induced retinitis </li></ul><ul><li>ICAM-1 (inflammatory cell adhesion molecule-1) antisense causes remission in 50% of Crohn's disease patients in clinical trials </li></ul>
  104. 106. Nuclear transport
  105. 107. Regulating mRNA Stability <ul><li>Information in 5  and 3  untranslated sequences important </li></ul><ul><li>Stability sequences increase half life (t 1/2 ) </li></ul><ul><li>Instability sequences decrease t 1/2 </li></ul><ul><ul><li>AUUUA rich sequences of ~50 bases (ARE) is bound by an ARE-binding protein </li></ul></ul><ul><ul><li>Causes mRNA to be deadenylated & lose PABP </li></ul></ul><ul><ul><li>Digested by poly(A) ribonuclease </li></ul></ul><ul><ul><li>Endonucleases digest RNA </li></ul></ul>
  106. 108. Decreased mRNA stability reduces amount of protein made <ul><li> and  tubulin levels demonstrate translational control </li></ul><ul><li>Add colchicine  microtubules dissociate </li></ul><ul><ul><li> and  subunit concentrations increase causing tubulin synthesis to drop </li></ul></ul><ul><li>Add vinblastine  microtubules dissociate and are precipitated </li></ul><ul><ul><li> and  subunit synthesis increases </li></ul></ul><ul><li>Difference probably caused by binding of free subunits to specific AA sequence encoded by 5  nucleotides </li></ul><ul><li>Protein-protein interaction activates an RNase that digests template </li></ul>
  107. 109. Altering mRNA Stability Allows for Translational Control
  108. 110. Prokaryotic Regulation Operons
  109. 111. Operons <ul><li>Units of transcription used to regulate gene expression in prokaryotes </li></ul><ul><li>Genes are grouped together in clusters for response to environmental conditions </li></ul><ul><li>Expression of cluster is regulated from one site </li></ul><ul><li>Inducible - are turned on in response to the presence of the substrate (the inducer) for a necessary enzyme </li></ul><ul><li>Repressible - presence of a specific molecule (the repressor) that inhibits gene expression </li></ul>
  110. 112. Activation of gene expression Recruitment of polymerase Allosteric activation
  111. 113. Cooperative binding and DNA bending activate gene expession
  112. 114. Negative Control Gene is expressed unless it is turned off by some regulatory molecule
  113. 115. Positive Control Gene only expressed if a regulatory molecule stimulates RNA synthesis
  114. 116. The lac Operon Prokaryotic genes do not have introns and exons  make polycistronic mRNA - continuous transcripts that are composed of many genes.
  115. 117. Expression of lac genes Control region
  116. 118. Negative Control
  117. 119. Repressor binds to operator but activator interacts with CTD tail
  118. 120. Inducer Changes Repressor Conformation
  119. 121. Operators
  120. 122. Lac Operon Has 3 Operators All 3 operators must be bound for maximal repression . Repressor binding to 2 operators causes a DNA conformational change  DNA bends away from the repressor forming a repression loop, preventing RNA polymerase access to the promoter
  121. 123. Operator Mutations Are Constitutive
  122. 124. lacI Mutations Are Constitutive
  123. 125. Repressor Mutants Are Super-repressed
  124. 126. Catabolite Repression
  125. 127. trp Operon <ul><li>Leader is composed of 162 nucleotides that contains another regulatory region, the attenuator </li></ul><ul><li>Tryptophan (Trp) is a co-repressor </li></ul><ul><li>When Trp binds to the normally inactive repressor protein , the repressor can bind to the operator and inhibit expression of the operon </li></ul>
  126. 128. trp operon is repressible
  127. 129. Transcription Occurs in the Absence of Tryptophan
  128. 130. Repressor Binds in the Presence of Tryptophan
  129. 131. Attenuation <ul><li>Attenuation only occurs in the presence of tryptophan </li></ul><ul><li>When the operon is repressed, transcription of the leader sequence is initiated but not completed </li></ul><ul><li>Transcription is attenuated 140 nucleotides into the leader sequence </li></ul><ul><li>Hairpin loop formed by the RNA encoded by the DNA in the attenuator region </li></ul><ul><li>Loop is followed by a polyU tract </li></ul>
  130. 132. Leader Sequence <ul><li>Leader encodes 2 triplets (UGG) that encode trp. </li></ul><ul><li>Leader also contains a translation initiation codon (AUG) </li></ul>
  131. 133. Attenuation Is Dependent Upon Leader Sequence
  132. 134. trp Leader Sequence
  133. 135. Tryptophan Available <ul><li>If trp is plentiful, charged trp-tRNA is present and translation occurs </li></ul><ul><li>Leader is made and the hairpin loop is formed </li></ul>
  134. 136. Tryptophan Present
  135. 137. Low Tryptophan Concentrations <ul><li>Charged trp-tRNA is not available and translation of the leader can not occur because the ribosome stalls </li></ul><ul><li>Ribosome stalling affects secondary structure of transcript  no hairpin loop is formed </li></ul><ul><li>Transcription of the structural genes proceeds </li></ul>
  136. 138. Low Tryptophan
  137. 139. Translation
  138. 140. tRNA <ul><li>Transcribed as one large primary transcript that is cleaved into smaller 4s tRNA molecules (70-90 nucleotides) </li></ul><ul><li>Have extensive post-trancriptional modifications </li></ul><ul><li>Have secondary and tertiary structure </li></ul><ul><li>32 different tRNAs due to wobble position </li></ul><ul><li>Nomenclature: Phenylalanyl tRNA = tRNA phe where phe is the cognate AA </li></ul>
  139. 141. tRNA contains rare nucleotides <ul><li>Rare nucleotides, i.e., pseudouridine, inosine, etc. </li></ul><ul><li>Some are observed in all tRNAs (dihyrouridine in D loop, pseudouridine (  ) in T  GC loop of acceptor stem, etc.), while others are specific for a particular tRNA or group of tRNAs. </li></ul>
  140. 142. tRNA exhibits atypical basepairing
  141. 143. tRNAs exhibit secondary and tertiary structure that results in the formation of loops and stems
  142. 144. Cloverleaf Model (Holley, 1965) <ul><li>Anticodon loop - binds to codon of mRNA </li></ul><ul><li>Acceptor stem - necessary for tRNA charging by tRNA synthetase. Places the terminus of the tRNA close to the active site of the enzyme </li></ul><ul><li>Amino acid (AA) binding site - contains the sequence 5  CCA 3  </li></ul><ul><li>Variable loop - also necessary for synthetase recognition </li></ul>
  143. 145. Aminoacyl tRNA Synthetases <ul><li>Very specific for AA and isoaccepting tRNA (cognate tRNA) </li></ul><ul><li>Cognate tRNA: multiple tRNAs that represent the same AA </li></ul><ul><li>Recognizing only one AA and tRNA is essential for the FIDELITY of the system b/c ribosome blindly accepts any charged tRNA with proper codon-anticodon interaction </li></ul><ul><li>Acceptor stem has a discriminator base at 3  acceptor end that is especially important for recognition specificity of a tRNA from 1 synthetase to another </li></ul><ul><li>Anticodon loop also contributes to discrimination </li></ul>
  144. 146. Synthetase Structure
  145. 147. Amino Acids
  146. 148. tRNA Charging <ul><li>Synthetase + AA +ATP  aminoacyl~adenylic acid-synthetase complex + PP i </li></ul><ul><li>Synthetase-aminoacyl~adenylic acid complex + tRNA  synthetase + charged tRNA + AMP </li></ul>
  147. 149. Chemistry of Charging <ul><li>Step one is an adenylylation: AA reacts with ATP, AMP transferred, PPi released </li></ul><ul><li>This step results in a high-energy ester bond joining the AA and AMP. </li></ul><ul><li>Breaking of this bond during peptidyl transferase reaction provides energy for formation of the peptide bond . </li></ul><ul><li>Step 2 is tRNA charging where AA reacts with tRNA </li></ul>
  148. 150. Recognition of correct tRNA
  149. 151. Biological polymerization of AA into polypeptide chains
  150. 152. Ribosome structure <ul><li>Composed of catalytic rRNA and structural proteins </li></ul><ul><li>Large and small subunits </li></ul><ul><li>rDNA mildly repetitive </li></ul><ul><li>Exists in clusters of tandem repeats (repeating sequences over and over) </li></ul><ul><li>All ribosomal RNA is transcribed as one large primary transcript followed by cleavage into smaller functional transcripts </li></ul>
  151. 153. Prokaryotic Ribosome <ul><li>Small subunit has </li></ul><ul><li>decoding center </li></ul><ul><li>Large subunit has peptidyl </li></ul><ul><li>transferase center </li></ul>
  152. 154. Prokaryotic vs. Eukaryotic Ribosome
  153. 155. Electron Micrographs of the Ribosome
  154. 156. Ribosome Active Sites
  155. 157. Mechanisms and Process
  156. 158. Transcription and translation are coupled in prokaryotes Overview Polyribosomes
  157. 159. Important Sequences for Initiation and Setting the Reading Frame <ul><li>Ribosome binding site (RBS) aka Shine-Delgarno sequence (-10): 5  AGGAGG 3  </li></ul><ul><li>Complementary to 16s rRNA sequence: 3  UCCUCC 5  </li></ul><ul><li>Start codon: 5  AUG 3  (sometimes GUG or UUG) </li></ul><ul><ul><li>Encodes initiator tRNA: fMet-tRNA i fMet = N-formyl methionine (different tRNA used for internal AUG) </li></ul></ul><ul><ul><li>Deformylase removes formyl group if Met is 1 st AA </li></ul></ul><ul><ul><li>Aminopeptidase removes Met if not 1 st AA </li></ul></ul>
  158. 160. Ribosome Has 2 Sites for Binding Charged tRNA
  159. 163. fMet-tRNA i fMet <ul><li>Has 3 GC pairs in stem before anticodon </li></ul><ul><li>loop that is necessary for entrance into P site </li></ul>
  160. 164. ONLY fMet-tRNA Can Enter the P Site <ul><li>Binding Sets the Reading Frame!!! </li></ul>
  161. 165. fMet Removed During Synthesis
  162. 166. Initiation
  163. 167. Initiation Factors <ul><li>Initiation factors (IF) absolutely required </li></ul><ul><li>Never observed in 70s ribosomal structure </li></ul><ul><li>All IFs released and GTP hydrolyzed so that 50s can bind </li></ul><ul><li>IF1 - stabilizes initiation complex </li></ul>
  164. 168. IF2 <ul><li>Essential for entry and binding of fmet-tRNA into P site </li></ul><ul><li>Binds GTP </li></ul><ul><li>Ribosome-dependent GTPase activity for formation of 70s ribosome </li></ul>
  165. 169. IF3 <ul><li>Stabilizes free 30s subunits </li></ul><ul><li>Prevents association of 50s </li></ul><ul><li>Dissociates ribosome into subunits at termination </li></ul>
  166. 170. <ul><li>IF3 occupies E site </li></ul><ul><li>IF1 binds to A site and IF2 binds to it leaving only P site open </li></ul><ul><li>fMet-tRNA i fMet binding is facilitated by interactions with IF2-bound GTP </li></ul><ul><li>When start codon and initiator base-pair, small subunit changes conformation and releases IF3 </li></ul><ul><li>Large subunit (50S) binds and stimulates IF2 GTPase activity, GTP hydrolyzed </li></ul><ul><li>IF2-GDP and IF1 released </li></ul><ul><li>70S ribosome formed allowing a charged tRNA to enter A site </li></ul>
  167. 171. Antibiotics Inhibit Translation
  168. 172. Eukaryotic Translation <ul><li>Translation initiation does not involve a Shine-Delgarno sequence </li></ul><ul><li>Kozak sequence (5  ACCAUGG 3  ) surrounds initiator codon </li></ul><ul><li>Ribosomes enter at the 5  cap and advance to the first AUG via small subunit linear scanning </li></ul><ul><li>Start recognized by anticodon of initiator tRNA which is why initiator is bound to small subunit prior to ribosome assembly </li></ul><ul><li>Control is usually exerted at the rate-limiting initiation step </li></ul>
  169. 173. 43s pre-initiation complex plus eIF4F/B bound mRNA form 48s initiation complex <ul><li>40s subunit binds to eIF1A + eIF3. </li></ul><ul><li>Next, eIF5B-GTP + eIF2  GTP  Met-tRNA i Met associate with small subunit and position initiator in P site </li></ul><ul><li>The cap-binding-protein complex = eIF4F finds the cap , acts as an RNA helicase to unwind 5  mRNA secondary structure </li></ul><ul><li>eIF4F has 3 subunits: </li></ul><ul><ul><li>A has RNA-dependent ATPase activity </li></ul></ul><ul><ul><li>E binds the cap </li></ul></ul><ul><ul><li>G is a docking site for the initiation complex - acts as the central adapter for the binding of regulation and initiation factors </li></ul></ul><ul><li>eIF4B activates helicase of eIF4F </li></ul><ul><li>eIF4F/B recruits 43s pre-initiation complex to mRNA via eIF3 = 48s initiation complex </li></ul>43s
  170. 174. Scanning to find the initiator <ul><li>Scanning is ATP-dependent and requires eIF4F to drive scan via its helicase activity </li></ul><ul><li>Find AUG & base-pair </li></ul><ul><li>eIF2, eIF3 and 4B released </li></ul><ul><li>Large subunit (60s) binds, stimulates eIF5B hydrolysis of bound GTP </li></ul><ul><li>5B-GDP & 1A released </li></ul><ul><li>Form 80s ribosome </li></ul>
  171. 175. Translation Requires Template Circularization <ul><li>eIF4F G subunit interacts directly with the poly(A) tail binding protein ( PABP ), and mRNA </li></ul><ul><li>When all of the initiation factors are bound, the mRNA template is circularized </li></ul><ul><li>Template circularization is thought to facilitate re-initiate translation </li></ul>
  172. 176. Translation Is Tightly Regulated <ul><li>Affinity of eIF4E for cap increased with phosphorylation </li></ul>MAP kinase interacting protein 1 PKC Preiss and Hentze, 1999 Current Opinion in Genetics & Development
  173. 177. eIF4E Binding Proteins Compete for Binding with eIF4G eIF4E, A and G = eIF4F complex Sonenberg & Gingras, 1998
  174. 178. Control at initiation by numerous signal transduction pathways Many signal transduction pathways converge on eIF4E and phosphorylate it as well as other IF factors & ribosomal proteins Sonenberg & Gingras, 1998 eIF, Devers, 1999
  175. 179. Elongation Factors <ul><li>EF-Tu </li></ul><ul><ul><li>Mediates entry of incoming aa-tRNA into A site </li></ul></ul><ul><ul><li>GTP is hydrolyzed after codon-anticodon recognition </li></ul></ul><ul><ul><li>Leaves after aa-tRNA is in A site </li></ul></ul><ul><ul><li>Does NOT recognize fmet-tRNA, only IF2 does </li></ul></ul><ul><ul><li>Function inhibited by the antibiotic kirromycin causing EF-Tu to remain bound to the ribosome </li></ul></ul><ul><ul><li>EF-Tu  GDP  inactive, can't bind aa-tRNA </li></ul></ul><ul><ul><li>EF-Tu  GTP  active, can bind aa-tRNA </li></ul></ul><ul><li>EF-Ts </li></ul><ul><ul><li>a GTPase exchange factor </li></ul></ul><ul><ul><li>Regenerates EF-Tu  GTP by displacing GDP from EF-Tu </li></ul></ul><ul><li>EF-G </li></ul><ul><ul><li>Stimulates translocation (movement of ribosome 3 nucleotides downstream) </li></ul></ul><ul><ul><li>Release requires GTP hydrolysis </li></ul></ul><ul><ul><li>EF-G  GTP regenerated from EF-G  GDP b/c GTP has higher affinity for EF-G than GDP does </li></ul></ul>
  176. 181. Elongation and the ribosome <ul><li>The ribosome is a ribozyme – the 23S rRNA (large subunit) catalyzes peptide bond formation </li></ul><ul><li>Ribosome very accurate – uses 3 mechanisms in addition to codon-anticodon interactions to select against incorrect codon-anticodon pairings: </li></ul><ul><li>Two adenine residues in 16s rRNA form tight interaction with minor groove of correct base pair. Don’t recognize non-Watson-Crick base pairs b/c form a minor groove they don’t recognize, significantly reducing affinity for mismatches. </li></ul><ul><li>Proofreading - One mismatch dramatically reduces GTPase activity of EF-Tu. Mechanism very similar to #1. </li></ul><ul><li>Accomodation – a form of proofreading that occurs after EF-Tu is released. tRNA is moved closer to peptidyl transferase center by rotation. Incorrectly paired tRNA will usually dissociate here. </li></ul>
  177. 183. Steps in Elongation <ul><li>EF-Tu  GTP binds to tRNA 3  end masking AA </li></ul><ul><li>EF-Tu  GTP  aa-tRNA binds to A site </li></ul><ul><li>Correct codon-anticodon match is made </li></ul><ul><li>Factor binding center activates Ef-Tu GTPase, GTP hydrolyzed, EF-Tu  GDP is released </li></ul><ul><li>Peptidyl transferase hydrolyzes bond between tRNA and AA yielding energy for peptide bond formation </li></ul><ul><li>Peptide bond formed between AAs in P site and A site and peptide transferred to A site </li></ul><ul><li>tRNA in P site is deacetylated and uncharged tRNA moves to E site </li></ul><ul><li>EF-G  GTP binds to A site on large subunit, contacts factor binding center </li></ul><ul><li>GTP hydrolyzed, ribosome translocation (ribosome moves 3 nucleotides 3  on mRNA)  A site empty, EF-G  GDP is released </li></ul><ul><li>Peptide chain emerges from tunnel in large subunit (after 30 aa are visible) </li></ul>
  178. 184. Overview EF-Tu carries aa-tRNA to A site
  179. 185. Peptide Bond Formation
  180. 186. Translocation
  181. 187. Translocation
  182. 188. Termination Factors <ul><li>Recognize stop codon, catalyze dissociation of ribosome </li></ul><ul><li>2 Class I Releasing Factors in prokaryotes (RF1 and 2) and 1 in eukaryotes (eRF1) </li></ul><ul><li>Class I RFs mimic tRNA and result in hydrolysis of the peptide chain from the tRNA in the P site </li></ul><ul><ul><li>Have a peptide anticodon that recognizes and interacts with the stop codon </li></ul></ul><ul><li>Class II RF stimulate release of Class I RFs and are regulated by GTP </li></ul><ul><ul><li>1 Class II RF in both prokaryotes and eukaryotes = RF3 & eRF3, respectively </li></ul></ul><ul><ul><li>Has high affinity for GDP NOT GTP </li></ul></ul><ul><li>Ribosome recycling factor (RRF) mimicks a tRNA in the A site and recruits EF-G </li></ul><ul><ul><li>Cooperates with IF3 and EF-G to remove tRNAs from E and P sites and release mRNA </li></ul></ul>
  183. 189. <ul><li>Steps </li></ul><ul><ul><li>RF1 (UAA or UAG) or RF2 (UAA or UGA) see stop codon </li></ul></ul><ul><ul><li>No aa-tRNA for stop codons so nothing in A site </li></ul></ul><ul><ul><li>RF1/2 mimic tRNA, activate ribosome to cleave peptide </li></ul></ul><ul><ul><li>RF3-GDP binds to ribosome, exchanges GDP for GTP causing release of RF1/2 </li></ul></ul><ul><ul><li>RF3-GTP interacts with the factor binding center of the ribosome causing GTP hydrolysis and RF3 release from the ribosome </li></ul></ul><ul><ul><li>RRF & EF-G cause tRNA to leave P & E sites </li></ul></ul><ul><ul><li>IF3 binds to small subunit causing dissociation of ribosome </li></ul></ul>
  184. 190. What happens if a ribosome stalls? <ul><li>A chimeric molecule called a tmRNA mimics tRNA and mRNA </li></ul><ul><li>For example, SsrA (charged with an alanine) can bind to EF-Tu-GDP, enter the A site and cause translocation and release of mRNA </li></ul><ul><li>In addition, a part of SsrA enters the mRNA channel of the ribosome and extends the ORF by 10 codons and a stop codon </li></ul><ul><li>This results in a protein with 10 extra AA that tag the protein as incomplete, causing cellular proteases to digest it </li></ul>
  185. 191. What does the cell do if there is an early stop codon? <ul><li>Normally, exon junction complexes (from splicing) are removed during translation </li></ul><ul><li>If a premature stop codon (a nonsense mutation) is encountered then the complexes still exist downstream from the mutation b/c translation stops before whole protein is made </li></ul><ul><li>This activates the nonsense mediated decay process where the remaining exon junction complexes recruit Upf proteins to the ribosome. Upf proteins activate the decapping enzyme that removes the 5  cap resulting in degradation of the mutated mRNA by 5   3  exonuclease. </li></ul>
  186. 192. What happens if there isn’t a stop codon? <ul><li>Nonstop mediated decay rescues the ribosome by recognizing that the ribosome has translated the poly-A + tail and stalled the ribosome </li></ul><ul><li>The stalled ribosome is bound by the exosome that includes Ski7, a protein related to eRF3, that causes ribosome dissociation and a 3  5  exonuclease that digests the mutated message </li></ul><ul><li>The poly-lysine tag on the carboxy terminus of the protein activates cellular proteases and the mutant protein is digested </li></ul>
  187. 193. A Translation Movie
  188. 194. Translation Animation Web Addresses