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Transcription and translation
 

Transcription and translation

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    Transcription and translation Transcription and translation Presentation Transcript

    • Prokaryotic Transcription
    • 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
    • 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
    • Phases of transcription
    • RNA Polymerase
    • RNA Polymerase
    • E. coli RNA Polymerase
      • Sigma subunit recognizes the transcription start site
      • Several different sigma subunits that recognize different promoters
    • 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)
    • 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
      =
    • Sigma 70 binding sites Region 3.2 acts as molecular mimic in abortive initiation; region 2.3 melts DNA
    • Open complex- note regions of binding by σ 70 binds to UP-element
    • The Transcription Unit
    • Transcription Occurs in a Bubble
    • Synthesis Occurs in the 5 ´ to 3´ Direction
    • Initiation
    •  
    • Transcription Requires Gyrase and Topoisomerase
    • 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
    • Intrinsic Termination
    • Rho-Dependent Termination
    • Antitermination
    • Eukaryotic Transcription
    • Regulation of Eukaryotic Gene Expression
    • 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
    • Translation of mRNA yields Protein
    • 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
    • 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
      • 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
    • A Typical Gene Transcribed by RNAP II
    • Anatomy of a Gene
    • Transcription Initiation Promoters
    • 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
    • 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
    • The Promoter Binds General Transcription Factors & RNAP II
    • In vitro Mutagenesis Shows Critical Sequences for Transcription Initiation
    • 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
      • 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
    • Enhancer Sequences (Response Elements)
    • Upstream Elements Vary with Gene Function
    • 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
    • Bending DNA Stimulates Transcription
    • 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.
      • 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)
    • General (Basal) TFs
    • 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
    • Stages of Initiation
    • 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 (?)
    • Assembling the Basal Complex
    • Initiation
    • TAFs Interact with TFIID (& TBP)
    • TBP-DNA TFIIB-TBP-DNA
    • 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
    • Mediator Complexes Are Composed of Many Coactivators
    • Coactivators Interact with TFs, But Do Not Bind to DNA
    • Mediators May Stabilize Pre-Initiation Complex After Chromatin Remodeling
      • Mediators associate with the CTD tail
      • Interact with DNA-bound activators
    •  
    • Multiple Pathways Affect Transcriptional Activation
    • 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
    • Promoter Escape Requires CTD Phosphorylation by TFIIH
    • Other Proteins Associated with Promoter Escape & Elongation
    • Elongation Phosphorylation of Ser 2 recruits splicing factors Phosphorylation of Ser 5 recruits capping factors Other factors include TFIIS, P-TEFb, TAT-SF1
    • 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
    • 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)
    • 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
    • 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
    • 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
    • EFs Modify Chromatin Structure
      • HMG14, FACT & Elongator modify and destabilize nucleosomes clearing the path for RNAP II movement
      • Elongator and SWI/SNF remodel chromatin
    • Elongation
    • Transcription Visualized Prokaryotic Eukaryotic
    • 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
    • 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
    • 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)
    • 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
    • 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
    • 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
    • Splicing Visualized Transcription initiated here Introns loop out as they are excised
    • Splicing Mechanisms Introns Are Classified by Their Splicing Mechanism
    • Splicing involves two transesterifications
    • Trans-Splicing joins exons from two different RNAs
    • 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
    • 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
    • hnRNPs Involved in Splicing Reactions
    • 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
    • Assembling the Splicesome
    • 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
    • Putting It All Together
    • Alternative Splicing Regulates Gene Expression
    • Alternative splicing
    • Alternative splicing results in families of proteins (splicing isoforms)
    • 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
    • Preprotachykinin (PPT) Gene P P
    • Splicing is regulated
    • 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
    • Exon shuffling
    • RNA Editing Changes the sequence of the RNA after transcription, but before translation
    • 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
    • gRNA Directs T. brucei RNA Editing
    • 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)
    • Apo-B Gene Is Modified by RNA Editing
    • 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
    • ADAR1 Mechanism
    • 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
    • RNAi/miRNA Mechanism Kosik Nature Reviews Neuroscience 7 , 911 – 920 (December 2006) | doi:10.1038/nrn2037
    • C. elegans makes lin-4 antisense to regulate lin-14 expression Now consider lin-4 antisense an miRNA
    • 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
    • Nuclear transport
    • 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
    • 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
    • Altering mRNA Stability Allows for Translational Control
    • Prokaryotic Regulation Operons
    • 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
    • Activation of gene expression Recruitment of polymerase Allosteric activation
    • Cooperative binding and DNA bending activate gene expession
    • Negative Control Gene is expressed unless it is turned off by some regulatory molecule
    • Positive Control Gene only expressed if a regulatory molecule stimulates RNA synthesis
    • The lac Operon Prokaryotic genes do not have introns and exons  make polycistronic mRNA - continuous transcripts that are composed of many genes.
    • Expression of lac genes Control region
    • Negative Control
    • Repressor binds to operator but activator interacts with CTD tail
    • Inducer Changes Repressor Conformation
    • Operators
    • 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
    • Operator Mutations Are Constitutive
    • lacI Mutations Are Constitutive
    • Repressor Mutants Are Super-repressed
    • Catabolite Repression
    • 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
    • trp operon is repressible
    • Transcription Occurs in the Absence of Tryptophan
    • Repressor Binds in the Presence of Tryptophan
    • 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
    • Leader Sequence
      • Leader encodes 2 triplets (UGG) that encode trp.
      • Leader also contains a translation initiation codon (AUG)
    • Attenuation Is Dependent Upon Leader Sequence
    • trp Leader Sequence
    • Tryptophan Available
      • If trp is plentiful, charged trp-tRNA is present and translation occurs
      • Leader is made and the hairpin loop is formed
    • Tryptophan Present
    • 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
    • Low Tryptophan
    • Translation
    • 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
    • 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.
    • tRNA exhibits atypical basepairing
    • tRNAs exhibit secondary and tertiary structure that results in the formation of loops and stems
    • 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
    • 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
    • Synthetase Structure
    • Amino Acids
    • tRNA Charging
      • Synthetase + AA +ATP  aminoacyl~adenylic acid-synthetase complex + PP i
      • Synthetase-aminoacyl~adenylic acid complex + tRNA  synthetase + charged tRNA + AMP
    • 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
    • Recognition of correct tRNA
    • Biological polymerization of AA into polypeptide chains
    • 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
    • Prokaryotic Ribosome
      • Small subunit has
      • decoding center
      • Large subunit has peptidyl
      • transferase center
    • Prokaryotic vs. Eukaryotic Ribosome
    • Electron Micrographs of the Ribosome
    • Ribosome Active Sites
    • Mechanisms and Process
    • Transcription and translation are coupled in prokaryotes Overview Polyribosomes
    • 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
    • Ribosome Has 2 Sites for Binding Charged tRNA
    •  
    •  
    • fMet-tRNA i fMet
      • Has 3 GC pairs in stem before anticodon
      • loop that is necessary for entrance into P site
    • ONLY fMet-tRNA Can Enter the P Site
      • Binding Sets the Reading Frame!!!
    • fMet Removed During Synthesis
    • Initiation
    • 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
    • IF2
      • Essential for entry and binding of fmet-tRNA into P site
      • Binds GTP
      • Ribosome-dependent GTPase activity for formation of 70s ribosome
    • IF3
      • Stabilizes free 30s subunits
      • Prevents association of 50s
      • Dissociates ribosome into subunits at termination
      • 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
    • Antibiotics Inhibit Translation
    • 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
    • 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
    • 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
    • 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
    • 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
    • eIF4E Binding Proteins Compete for Binding with eIF4G eIF4E, A and G = eIF4F complex Sonenberg & Gingras, 1998
    • 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
    • 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
    •  
    • 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.
    •  
    • 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)
    • Overview EF-Tu carries aa-tRNA to A site
    • Peptide Bond Formation
    • Translocation
    • Translocation
    • 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
      • 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
    • 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
    • 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.
    • 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
    • A Translation Movie
    • 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