Transcription and translation

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  • 1. Prokaryotic Transcription
  • 2. Transcription
    • DNA-dependent RNA synthesis
    • RNA polymerase doesn’t require a primer
    • Ribonucleotides not deoxyribonucleotides incorporated into the polymer
    • Uracil substituted for thymine
    • Template for transcription is the antisense strand
  • 3. Stages
    • Initiation : Occurs after promoter recognition and polymerase binding when the first rNTP is inserted
    • Elongation : adding rNTPs to chain
      • Sigma subunit dissociates after a few bases are added to the chain
    • Termination : dissociation of core polymerase and release of RNA transcript
  • 4. Phases of transcription
  • 5. RNA Polymerase
  • 6. RNA Polymerase
  • 7. E. coli RNA Polymerase
    • Sigma subunit recognizes the transcription start site
    • Several different sigma subunits that recognize different promoters
  • 8. Promoters
    • Sequences that regulate the efficiency of transcription initiation
    • Can be strong or weak
    • Contain palindromic (e.g. RADAR) consensus sequences recognized by sigma subunit
    • TTGACA – sigma 70 subunit recognition domain is always at -35
    • TATAAT - Pribnow (or TATA) box  always at -10 for unwinding of helix
    • Distance between TATAAT and TTGACA very important for polymerase binding (~17 bp)
  • 9. Prokaryotic Promoters
    • -35 and 10 regions recognized by regions 2 & 4 of σ 70 factor
    • extended -10 recognized by region 3
    • Region 4 has helix-turn-helix DNA binding motif
      • 1 helix interacts with major groove at -35 & other lies on top of groove interacting with bases
      •  helix interacts with -10 region on nontemplate strand
      • UP-element recognized by  CTD
    =
  • 10. Sigma 70 binding sites Region 3.2 acts as molecular mimic in abortive initiation; region 2.3 melts DNA
  • 11. Open complex- note regions of binding by σ 70 binds to UP-element
  • 12. The Transcription Unit
  • 13. Transcription Occurs in a Bubble
  • 14. Synthesis Occurs in the 5 ´ to 3´ Direction
  • 15. Initiation
  • 16.  
  • 17. Transcription Requires Gyrase and Topoisomerase
  • 18. Termination
    • Intrinsic termination - caused by a palindromic sequence in the DNA template that results in the formation of a hairpin loop that prevents elongation
    • Rho-dependent termination -  protein physically interacts with RNA transcript preventing elongation. Most gene transcription is terminated this way in prokaryotes
    • Antitermination - viral protein allows polymerase to read through the termination sequence making a different protein
  • 19. Intrinsic Termination
  • 20. Rho-Dependent Termination
  • 21. Antitermination
  • 22. Eukaryotic Transcription
  • 23. Regulation of Eukaryotic Gene Expression
  • 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
  • 25. Translation of mRNA yields Protein
  • 26. Eukaryotes Have 3 RNA Polymerases
    • Pol I synthesizes rRNA in the nucleolus, not inhibited by  -amanitin (octapeptide synthesized by a mushroom)
    • Pol II synthesizes mRNA and snRNA, inhibited by low concentrations of  -amanitin
    • Pol III synthesizes 5s rRNA and tRNA, inhibited by high concentrations of  -amanitin
  • 27. RNA Polymerase II
    • 12 subunits shaped like a crab claw
    • Jaws grip template & clamp locks template at catalytic site for high processivity
    • 1 Mg 2+ at catalytic site
    • 8-9 bp of hybrid puts 3 ´ OH at catalytic site
    • 20 bp DNA downstream in cleft
    • RNA fits in grooves
  • 28.
    • Several channels lead to the active site
    • 2 DNA channels up and downstream from transcription bubble make DNA bend about 90°
    • Tunnel on opposite side of DNA entry for NTP diffusion and incorporation into RNA
    • Rudder protrudes from active site to split RNA-DNA hybrid
    • RNA exits from another channel opposite
    • DNA entry that has a protein flap that may aid in elongation and termination
  • 29. A Typical Gene Transcribed by RNAP II
  • 30. Anatomy of a Gene
  • 31. Transcription Initiation Promoters
  • 32. Overview
    • Elements in the promoter can be common to all genes and used constitutively
    • Other elements are gene specific: identify particular classes of genes
    • These elements exist in different combinations in individual genes
    • Housekeeping genes contain elements recognized by general and upstream factors and are transcribed in all cells
  • 33. Cis-acting Elements Located at a Fixed Distance from Initiation Site
    • General elements bound by basal factors for initiation are called Consensus Sequences
    • GC box: -110 = GGGCGG, often multiple copies
    • CAAT box: -80 = GGCCAATCT
    • TATA Box: -30 (Goldberg-Hogness Box) = TATAAAA is pretty nonspecific, fixes initiation site because it is easily denatured
    • RNAP II binds at TATA Box
  • 34. The Promoter Binds General Transcription Factors & RNAP II
  • 35. In vitro Mutagenesis Shows Critical Sequences for Transcription Initiation
  • 36. Enhancers
    • DNA sequences that can modulate transcription from a distance
    • Can be upstream, close to start, or down stream of start site
    • Aren't always directly involved in template binding, but are essential to efficient transcription
    • Can be negative or positive, but are usually positive
    • Position is not fixed - can be upstream, downstream or within an intron
  • 37.
    • Can be removed and put back into a different gene and work
    • Can be inverted with no effect on activity
    • Control chromatin structure and rate of transcription (affect efficiency and stimulate)
    • Not necessary for transcription but are necessary for full activation (basal vs. induced expression)
    • Are responsible for time and tissue-specific gene expression
    • Interact with regulatory proteins & transcription factors
  • 38. Enhancer Sequences (Response Elements)
  • 39. Upstream Elements Vary with Gene Function
  • 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
  • 41. Bending DNA Stimulates Transcription
  • 42. Transcription Factors
    • Trans-acting factors
    • Directly facilitate template binding
    • 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.
    • The "  factor" for eukaryotes.
    • Different transcription factors may compete for promoter/ binding elements, some of which overlap.
    • Concentration and affinity effect which binds.
    • Sometimes same binding element binds different transcriptional factors in a tissue specific manner.
    • Some are gene specific.
  • 43.
    • Basal Factors = TFII s - always associated with Pol II
    • True Activators : modular proteins with 2 domains
    • DNA-Binding Domain : DNA-Protein Interaction
      • Distinct structural motifs
      • DNA-Binding Domains are classified by structural motif
    • Trans-activating Domain : Protein-Protein Interaction
    • Can interact with RNAP II or other transcription factors at the promoter and coactivators (hormones, small metabolites)
  • 44. General (Basal) TFs
  • 45. Pol II Core Promoter
    • Core promoter – minimal set of sequences necessary for accurate initiation
    • BRE – TFIIB recognition element
    • TATA Box
    • Inr- Initiator
    • DPE – Downstream element
    • Typical promoter usually include only 2 or 3 of any of these
    • Upstream lie regulatory elements
  • 46. Stages of Initiation
  • 47. Commitment
    • TFII D complex binds to TATA box via TATA Box Binding Protein (TBP) and TAF's (TATA Associated Factors) ~20 bases involved
    • TFII D complex contacts DNA - changes conformation to facilitate binding of TFII B and A
    • RNAP II complexed to TFII F binds next
    • TFII E
    • TFII H (helicase & kinase activity)
    • NTPs enter
    • TFII J (?)
  • 48. Assembling the Basal Complex
  • 49. Initiation
  • 50. TAFs Interact with TFIID (& TBP)
  • 51. TBP-DNA TFIIB-TBP-DNA
  • 52. Mediator Complexes
    • Multiprotein complexes associated with RNAP II
    • Do not bind to DNA
    • Act like control panels for RNAP II
    • Mediate interactions with TFs
    • Often required for the function of TFs
    • Integrate all of the positive and negative regulatory signals for RNAP II and "determine" how much message should be made.
    • Probably interact with the C-terminal domain (CTD) of the largest RNAP II subunit
  • 53. Mediator Complexes Are Composed of Many Coactivators
  • 54. Coactivators Interact with TFs, But Do Not Bind to DNA
  • 55. Mediators May Stabilize Pre-Initiation Complex After Chromatin Remodeling
    • Mediators associate with the CTD tail
    • Interact with DNA-bound activators
  • 56.  
  • 57. Multiple Pathways Affect Transcriptional Activation
  • 58. Promoter Escape
    • Pol II moves away from the promoter
    • Synthesizes 10-15 nucleotides
    • Dissociates from general initiation factors
    • Cannot occur unless CTD is hyperphosphorylated by TFII H
  • 59. Promoter Escape Requires CTD Phosphorylation by TFIIH
  • 60. Other Proteins Associated with Promoter Escape & Elongation
  • 61. Elongation Phosphorylation of Ser 2 recruits splicing factors Phosphorylation of Ser 5 recruits capping factors Other factors include TFIIS, P-TEFb, TAT-SF1
  • 62. Abortive Transcriptionand Proofreading
    • Transcripts smaller than about 9 nucleotides are aborted
    • TFII F acts to decrease abortive transcription (by increasing rate of polymerization?)
    • TFIIS contributes to Pol II’s proofreading by stimulating its inherent RNase activity
  • 63. Arrested Transcription
    • Transcription can also be arrested at promoter escape, potentially by TFII F binding to promoter ahead of transcriptional start site
    • 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
    • TFII H also recognizes damaged template DNA and recruits proteins for DNA excision-repair
    • 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)
  • 64. EFs Can Reactivate Arrested RNAP II
    • 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
  • 65. EFs Can Prevent RNAP II Arrest
    • P-TEFb is a cyclin-dependent kinase that phosphorylates CTD to prevent elongation arrest
    • DSIF and NELF are negative regulators of elongation
      • Both interact with Pol II in its hypophosphorylated form
      • DSIF/NELF blockade is removed by P-TEFb phosphorylation of CTD
  • 66. RNAP II Pausing Is the Rate-limiting Step in Elongation
    • EFs can prevent pausing
    • TFII F, ELL, Elongin & CSB suppress pausing by decreasing the time RNAP II spends in an inactive conformation increasing rate the of transcription
  • 67. EFs Modify Chromatin Structure
    • HMG14, FACT & Elongator modify and destabilize nucleosomes clearing the path for RNAP II movement
    • Elongator and SWI/SNF remodel chromatin
  • 68. Elongation
  • 69. Transcription Visualized Prokaryotic Eukaryotic
  • 70. RNA processing is coupled to elongation
    • RNAP II CTD interacts with RNA processing proteins to process the transcript as it comes through the flap at the end of tunnel
    • 7-methyl guanine cap, splicing and polyadenylation are coupled to elongation
  • 71. Capping the 5 ´ end of the transcript
    • The 7-methyl guanosine cap is added to the 5´-PO 4 before the transcript is 30 nucleotides long
    • May be used to attenuate mRNA output
    • Unique 5  -5  bond is added shortly after transcription initiation via 3 reactions
  • 72. Capping reactions
    • Phosphatase removes 5  phosphate
    • Guanylyl transferase catalyzes a condensation between the 5  triphosphate and GTP
    • Guanine-7-methyltransferase transfers the methyl group
    • Ser 5 of CTD dephosphorylated and capping machinery leaves
    • All eukaryotes possess a methyl on N7 of the terminal guanine
    • Higher eukaryotes often add a second methyl group to the penultimate base at the 2  -O position (2  -O-methyltransferase)
  • 73. 3  Polyadenylation
    • Ser 2 must be phosphorylated for poly(A) factor recruitment
    • Length of poly(A) tail determined by proteins bound to poly(A) sequence
    • AAUAA signals the addition of the poly(A) tail
    • Poly(A) polymerase adds ~200 A residues to the free 3  -OH of the transcript
    • Poly(A) tail leads to cleavage ~10-35 upstream of signal
    • Cleavage polyadenylation stimulatory factor (CPSF) recognizes the polyadenylation sequence (AAUAAA)
      • associates with TFIID first, then jumps on to CTD after initiation
    • Cleavage stimulatory factor (CstF) also interacts with CTD
      • necessary for elongation
  • 74. Poly(A) Tail Confers Stability to mRNA
    • Poly(A) tail is associated with the poly(A)-binding protein (PABP)
    • Poly(A) tail + PABP thought to confer stability to many mRNA transcripts and is involved in translation initiation
  • 75. RNA Splicing
    • 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
    • Work in Phil Sharp’s lab at MIT by his post-doc Sarah Flint demonstrated that eukaryotic gene structure differed from prokaryotic gene structure
    • Walter Gilbert named these gene regions:
      • Exon = expressed sequences
      • Intron = intervening sequences
  • 76. Splicing Visualized Transcription initiated here Introns loop out as they are excised
  • 77. Splicing Mechanisms Introns Are Classified by Their Splicing Mechanism
  • 78. Splicing involves two transesterifications
  • 79. Trans-Splicing joins exons from two different RNAs
  • 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 "New" 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
  • 81. Spliceosome
    • snRNA s mall n uclear RNAs
    • snRNPs small ribonucleoproteins (snurps)
      • rich in uridine
      • only in the nucleus
      • designated U1, U2, etc.
    • Serine-Arginine (SR) proteins act as bridging factors
      • N- terminal RNA recognition motifs for binding hnRNA
      • C-terminal arg-ser (RS) rich sequences for protein-protein interactions with RS domains in snurps
  • 82. hnRNPs Involved in Splicing Reactions
  • 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
  • 84. Assembling the Splicesome
  • 85. Splicing and errors
    • Errors are decreased by coupling transcription and splicing – see 3 ′ site as transcribed so no competition from other sites
    • Errors decreased by exonic splicing enhancers – ser arg rich sites that are bound by the essential SR proteins that recruit snurps to splice sites
    • SR proteins also necessary for alternative splicing
  • 86. Putting It All Together
  • 87. Alternative Splicing Regulates Gene Expression
  • 88. Alternative splicing
  • 89. Alternative splicing results in families of proteins (splicing isoforms)
  • 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
  • 91. Preprotachykinin (PPT) Gene P P
  • 92. Splicing is regulated
  • 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
  • 94. Exon shuffling
  • 95. RNA Editing Changes the sequence of the RNA after transcription, but before translation
  • 96. Insertion/Deletion Editing
    • Nucleotide addtion or subtraction directed by guide RNA (gRNA) templates
    • Add poly(U) to form initiation codon and set reading frame
    • gRNA template complementary to edited region of final RNA transcript
    • gRNA base pairs with pre-RNA and directs editing complex to make appropriate changes to RNA transcript
  • 97. gRNA Directs T. brucei RNA Editing
  • 98. Substitution editing
    • Nucleotides are altered by substituting one for another
    • Prevalent in mitochondria and chloroplasts
    • Apolipoprotein B (apo B) exists in long and short forms
    • Intestine: protein complex binds to "mooring" sequence downstream of editing site
    • C to U substitution: CAA = glutamine; UAA = stop (short form)
  • 99. Apo-B Gene Is Modified by RNA Editing
  • 100. Transcription-Induced Z-DNA, dsRNA & RNA Editing
    • Z-DNA stabilized by negative supercoiling induced by RNAP II
    • dsRNA editing substrate forms by 3 ´ intron folding back on exon to be edited
    • A denosine D eaminase A cting on R NA (ADAR) 1 Binds to dsRNA and Z-DNA
    • It is proposed that binding to Z-DNA allosterically activates ADAR1
    • ADAR1 deaminates adenosine to inosine
    • I read as G during translation resulting in glutamine (CAG) to arginine (CGG) substitution
    • Occurs in Glutamine Receptor-B and Serotonin-2C receptor
  • 101. ADAR1 Mechanism
  • 102. Antisense (RNAi, siRNA and miRNA) Regulation of Translation
    • All use a large dsRNA that activates an enzyme called dicer
    • Dicer digests large transcript into short pieces (21-23 nt) that recruit the RISC complex of proteins
    • 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
  • 103. RNAi/miRNA Mechanism Kosik Nature Reviews Neuroscience 7 , 911 – 920 (December 2006) | doi:10.1038/nrn2037
  • 104. C. elegans makes lin-4 antisense to regulate lin-14 expression Now consider lin-4 antisense an miRNA
  • 105. Have Many Antisense "Drugs" in Clinical Trials
    • Vitravene  is an antisense “ drug ” that targets cytomeglavirus in AIDS patients with cytomeglavirus-induced retinitis
    • ICAM-1 (inflammatory cell adhesion molecule-1) antisense causes remission in 50% of Crohn's disease patients in clinical trials
  • 106. Nuclear transport
  • 107. Regulating mRNA Stability
    • Information in 5  and 3  untranslated sequences important
    • Stability sequences increase half life (t 1/2 )
    • Instability sequences decrease t 1/2
      • AUUUA rich sequences of ~50 bases (ARE) is bound by an ARE-binding protein
      • Causes mRNA to be deadenylated & lose PABP
      • Digested by poly(A) ribonuclease
      • Endonucleases digest RNA
  • 108. Decreased mRNA stability reduces amount of protein made
    •  and  tubulin levels demonstrate translational control
    • Add colchicine  microtubules dissociate
      •  and  subunit concentrations increase causing tubulin synthesis to drop
    • Add vinblastine  microtubules dissociate and are precipitated
      •  and  subunit synthesis increases
    • Difference probably caused by binding of free subunits to specific AA sequence encoded by 5  nucleotides
    • Protein-protein interaction activates an RNase that digests template
  • 109. Altering mRNA Stability Allows for Translational Control
  • 110. Prokaryotic Regulation Operons
  • 111. Operons
    • Units of transcription used to regulate gene expression in prokaryotes
    • Genes are grouped together in clusters for response to environmental conditions
    • Expression of cluster is regulated from one site
    • Inducible - are turned on in response to the presence of the substrate (the inducer) for a necessary enzyme
    • Repressible - presence of a specific molecule (the repressor) that inhibits gene expression
  • 112. Activation of gene expression Recruitment of polymerase Allosteric activation
  • 113. Cooperative binding and DNA bending activate gene expession
  • 114. Negative Control Gene is expressed unless it is turned off by some regulatory molecule
  • 115. Positive Control Gene only expressed if a regulatory molecule stimulates RNA synthesis
  • 116. The lac Operon Prokaryotic genes do not have introns and exons  make polycistronic mRNA - continuous transcripts that are composed of many genes.
  • 117. Expression of lac genes Control region
  • 118. Negative Control
  • 119. Repressor binds to operator but activator interacts with CTD tail
  • 120. Inducer Changes Repressor Conformation
  • 121. Operators
  • 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
  • 123. Operator Mutations Are Constitutive
  • 124. lacI Mutations Are Constitutive
  • 125. Repressor Mutants Are Super-repressed
  • 126. Catabolite Repression
  • 127. trp Operon
    • Leader is composed of 162 nucleotides that contains another regulatory region, the attenuator
    • Tryptophan (Trp) is a co-repressor
    • When Trp binds to the normally inactive repressor protein , the repressor can bind to the operator and inhibit expression of the operon
  • 128. trp operon is repressible
  • 129. Transcription Occurs in the Absence of Tryptophan
  • 130. Repressor Binds in the Presence of Tryptophan
  • 131. Attenuation
    • Attenuation only occurs in the presence of tryptophan
    • When the operon is repressed, transcription of the leader sequence is initiated but not completed
    • Transcription is attenuated 140 nucleotides into the leader sequence
    • Hairpin loop formed by the RNA encoded by the DNA in the attenuator region
    • Loop is followed by a polyU tract
  • 132. Leader Sequence
    • Leader encodes 2 triplets (UGG) that encode trp.
    • Leader also contains a translation initiation codon (AUG)
  • 133. Attenuation Is Dependent Upon Leader Sequence
  • 134. trp Leader Sequence
  • 135. Tryptophan Available
    • If trp is plentiful, charged trp-tRNA is present and translation occurs
    • Leader is made and the hairpin loop is formed
  • 136. Tryptophan Present
  • 137. Low Tryptophan Concentrations
    • Charged trp-tRNA is not available and translation of the leader can not occur because the ribosome stalls
    • Ribosome stalling affects secondary structure of transcript  no hairpin loop is formed
    • Transcription of the structural genes proceeds
  • 138. Low Tryptophan
  • 139. Translation
  • 140. tRNA
    • Transcribed as one large primary transcript that is cleaved into smaller 4s tRNA molecules (70-90 nucleotides)
    • Have extensive post-trancriptional modifications
    • Have secondary and tertiary structure
    • 32 different tRNAs due to wobble position
    • Nomenclature: Phenylalanyl tRNA = tRNA phe where phe is the cognate AA
  • 141. tRNA contains rare nucleotides
    • Rare nucleotides, i.e., pseudouridine, inosine, etc.
    • 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.
  • 142. tRNA exhibits atypical basepairing
  • 143. tRNAs exhibit secondary and tertiary structure that results in the formation of loops and stems
  • 144. Cloverleaf Model (Holley, 1965)
    • Anticodon loop - binds to codon of mRNA
    • Acceptor stem - necessary for tRNA charging by tRNA synthetase. Places the terminus of the tRNA close to the active site of the enzyme
    • Amino acid (AA) binding site - contains the sequence 5  CCA 3 
    • Variable loop - also necessary for synthetase recognition
  • 145. Aminoacyl tRNA Synthetases
    • Very specific for AA and isoaccepting tRNA (cognate tRNA)
    • Cognate tRNA: multiple tRNAs that represent the same AA
    • 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
    • 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
    • Anticodon loop also contributes to discrimination
  • 146. Synthetase Structure
  • 147. Amino Acids
  • 148. tRNA Charging
    • Synthetase + AA +ATP  aminoacyl~adenylic acid-synthetase complex + PP i
    • Synthetase-aminoacyl~adenylic acid complex + tRNA  synthetase + charged tRNA + AMP
  • 149. Chemistry of Charging
    • Step one is an adenylylation: AA reacts with ATP, AMP transferred, PPi released
    • This step results in a high-energy ester bond joining the AA and AMP.
    • Breaking of this bond during peptidyl transferase reaction provides energy for formation of the peptide bond .
    • Step 2 is tRNA charging where AA reacts with tRNA
  • 150. Recognition of correct tRNA
  • 151. Biological polymerization of AA into polypeptide chains
  • 152. Ribosome structure
    • Composed of catalytic rRNA and structural proteins
    • Large and small subunits
    • rDNA mildly repetitive
    • Exists in clusters of tandem repeats (repeating sequences over and over)
    • All ribosomal RNA is transcribed as one large primary transcript followed by cleavage into smaller functional transcripts
  • 153. Prokaryotic Ribosome
    • Small subunit has
    • decoding center
    • Large subunit has peptidyl
    • transferase center
  • 154. Prokaryotic vs. Eukaryotic Ribosome
  • 155. Electron Micrographs of the Ribosome
  • 156. Ribosome Active Sites
  • 157. Mechanisms and Process
  • 158. Transcription and translation are coupled in prokaryotes Overview Polyribosomes
  • 159. Important Sequences for Initiation and Setting the Reading Frame
    • Ribosome binding site (RBS) aka Shine-Delgarno sequence (-10): 5  AGGAGG 3 
    • Complementary to 16s rRNA sequence: 3  UCCUCC 5 
    • Start codon: 5  AUG 3  (sometimes GUG or UUG)
      • Encodes initiator tRNA: fMet-tRNA i fMet = N-formyl methionine (different tRNA used for internal AUG)
      • Deformylase removes formyl group if Met is 1 st AA
      • Aminopeptidase removes Met if not 1 st AA
  • 160. Ribosome Has 2 Sites for Binding Charged tRNA
  • 161.  
  • 162.  
  • 163. fMet-tRNA i fMet
    • Has 3 GC pairs in stem before anticodon
    • loop that is necessary for entrance into P site
  • 164. ONLY fMet-tRNA Can Enter the P Site
    • Binding Sets the Reading Frame!!!
  • 165. fMet Removed During Synthesis
  • 166. Initiation
  • 167. Initiation Factors
    • Initiation factors (IF) absolutely required
    • Never observed in 70s ribosomal structure
    • All IFs released and GTP hydrolyzed so that 50s can bind
    • IF1 - stabilizes initiation complex
  • 168. IF2
    • Essential for entry and binding of fmet-tRNA into P site
    • Binds GTP
    • Ribosome-dependent GTPase activity for formation of 70s ribosome
  • 169. IF3
    • Stabilizes free 30s subunits
    • Prevents association of 50s
    • Dissociates ribosome into subunits at termination
  • 170.
    • IF3 occupies E site
    • IF1 binds to A site and IF2 binds to it leaving only P site open
    • fMet-tRNA i fMet binding is facilitated by interactions with IF2-bound GTP
    • When start codon and initiator base-pair, small subunit changes conformation and releases IF3
    • Large subunit (50S) binds and stimulates IF2 GTPase activity, GTP hydrolyzed
    • IF2-GDP and IF1 released
    • 70S ribosome formed allowing a charged tRNA to enter A site
  • 171. Antibiotics Inhibit Translation
  • 172. Eukaryotic Translation
    • Translation initiation does not involve a Shine-Delgarno sequence
    • Kozak sequence (5  ACCAUGG 3  ) surrounds initiator codon
    • Ribosomes enter at the 5  cap and advance to the first AUG via small subunit linear scanning
    • Start recognized by anticodon of initiator tRNA which is why initiator is bound to small subunit prior to ribosome assembly
    • Control is usually exerted at the rate-limiting initiation step
  • 173. 43s pre-initiation complex plus eIF4F/B bound mRNA form 48s initiation complex
    • 40s subunit binds to eIF1A + eIF3.
    • Next, eIF5B-GTP + eIF2  GTP  Met-tRNA i Met associate with small subunit and position initiator in P site
    • The cap-binding-protein complex = eIF4F finds the cap , acts as an RNA helicase to unwind 5  mRNA secondary structure
    • eIF4F has 3 subunits:
      • A has RNA-dependent ATPase activity
      • E binds the cap
      • G is a docking site for the initiation complex - acts as the central adapter for the binding of regulation and initiation factors
    • eIF4B activates helicase of eIF4F
    • eIF4F/B recruits 43s pre-initiation complex to mRNA via eIF3 = 48s initiation complex
    43s
  • 174. Scanning to find the initiator
    • Scanning is ATP-dependent and requires eIF4F to drive scan via its helicase activity
    • Find AUG & base-pair
    • eIF2, eIF3 and 4B released
    • Large subunit (60s) binds, stimulates eIF5B hydrolysis of bound GTP
    • 5B-GDP & 1A released
    • Form 80s ribosome
  • 175. Translation Requires Template Circularization
    • eIF4F G subunit interacts directly with the poly(A) tail binding protein ( PABP ), and mRNA
    • When all of the initiation factors are bound, the mRNA template is circularized
    • Template circularization is thought to facilitate re-initiate translation
  • 176. Translation Is Tightly Regulated
    • Affinity of eIF4E for cap increased with phosphorylation
    MAP kinase interacting protein 1 PKC Preiss and Hentze, 1999 Current Opinion in Genetics & Development
  • 177. eIF4E Binding Proteins Compete for Binding with eIF4G eIF4E, A and G = eIF4F complex Sonenberg & Gingras, 1998
  • 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
  • 179. Elongation Factors
    • EF-Tu
      • Mediates entry of incoming aa-tRNA into A site
      • GTP is hydrolyzed after codon-anticodon recognition
      • Leaves after aa-tRNA is in A site
      • Does NOT recognize fmet-tRNA, only IF2 does
      • Function inhibited by the antibiotic kirromycin causing EF-Tu to remain bound to the ribosome
      • EF-Tu  GDP  inactive, can't bind aa-tRNA
      • EF-Tu  GTP  active, can bind aa-tRNA
    • EF-Ts
      • a GTPase exchange factor
      • Regenerates EF-Tu  GTP by displacing GDP from EF-Tu
    • EF-G
      • Stimulates translocation (movement of ribosome 3 nucleotides downstream)
      • Release requires GTP hydrolysis
      • EF-G  GTP regenerated from EF-G  GDP b/c GTP has higher affinity for EF-G than GDP does
  • 180.  
  • 181. Elongation and the ribosome
    • The ribosome is a ribozyme – the 23S rRNA (large subunit) catalyzes peptide bond formation
    • Ribosome very accurate – uses 3 mechanisms in addition to codon-anticodon interactions to select against incorrect codon-anticodon pairings:
    • 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.
    • Proofreading - One mismatch dramatically reduces GTPase activity of EF-Tu. Mechanism very similar to #1.
    • 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.
  • 182.  
  • 183. Steps in Elongation
    • EF-Tu  GTP binds to tRNA 3  end masking AA
    • EF-Tu  GTP  aa-tRNA binds to A site
    • Correct codon-anticodon match is made
    • Factor binding center activates Ef-Tu GTPase, GTP hydrolyzed, EF-Tu  GDP is released
    • Peptidyl transferase hydrolyzes bond between tRNA and AA yielding energy for peptide bond formation
    • Peptide bond formed between AAs in P site and A site and peptide transferred to A site
    • tRNA in P site is deacetylated and uncharged tRNA moves to E site
    • EF-G  GTP binds to A site on large subunit, contacts factor binding center
    • GTP hydrolyzed, ribosome translocation (ribosome moves 3 nucleotides 3  on mRNA)  A site empty, EF-G  GDP is released
    • Peptide chain emerges from tunnel in large subunit (after 30 aa are visible)
  • 184. Overview EF-Tu carries aa-tRNA to A site
  • 185. Peptide Bond Formation
  • 186. Translocation
  • 187. Translocation
  • 188. Termination Factors
    • Recognize stop codon, catalyze dissociation of ribosome
    • 2 Class I Releasing Factors in prokaryotes (RF1 and 2) and 1 in eukaryotes (eRF1)
    • Class I RFs mimic tRNA and result in hydrolysis of the peptide chain from the tRNA in the P site
      • Have a peptide anticodon that recognizes and interacts with the stop codon
    • Class II RF stimulate release of Class I RFs and are regulated by GTP
      • 1 Class II RF in both prokaryotes and eukaryotes = RF3 & eRF3, respectively
      • Has high affinity for GDP NOT GTP
    • Ribosome recycling factor (RRF) mimicks a tRNA in the A site and recruits EF-G
      • Cooperates with IF3 and EF-G to remove tRNAs from E and P sites and release mRNA
  • 189.
    • Steps
      • RF1 (UAA or UAG) or RF2 (UAA or UGA) see stop codon
      • No aa-tRNA for stop codons so nothing in A site
      • RF1/2 mimic tRNA, activate ribosome to cleave peptide
      • RF3-GDP binds to ribosome, exchanges GDP for GTP causing release of RF1/2
      • RF3-GTP interacts with the factor binding center of the ribosome causing GTP hydrolysis and RF3 release from the ribosome
      • RRF & EF-G cause tRNA to leave P & E sites
      • IF3 binds to small subunit causing dissociation of ribosome
  • 190. What happens if a ribosome stalls?
    • A chimeric molecule called a tmRNA mimics tRNA and mRNA
    • For example, SsrA (charged with an alanine) can bind to EF-Tu-GDP, enter the A site and cause translocation and release of mRNA
    • In addition, a part of SsrA enters the mRNA channel of the ribosome and extends the ORF by 10 codons and a stop codon
    • This results in a protein with 10 extra AA that tag the protein as incomplete, causing cellular proteases to digest it
  • 191. What does the cell do if there is an early stop codon?
    • Normally, exon junction complexes (from splicing) are removed during translation
    • 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
    • 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.
  • 192. What happens if there isn’t a stop codon?
    • Nonstop mediated decay rescues the ribosome by recognizing that the ribosome has translated the poly-A + tail and stalled the ribosome
    • 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
    • The poly-lysine tag on the carboxy terminus of the protein activates cellular proteases and the mutant protein is digested
  • 193. A Translation Movie
  • 194. Translation Animation Web Addresses http://www.geocities.com/CapeCanaveral/Lab/5451/transgif.htm http://tidepool.st.usm.edu/crswr/protsynthmov.html http://www.bio.cmu.edu/Courses/BiochemMols/ribosome/70S.htm http://www.ncc.gmu.edu/dna/ANIMPROT.htm