Translation

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Translation

• Chapter 27

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Central Dogma

• Genetic code contained w/in seqs of 4
  deoxynucleotide bases of DNA
• 1 gene ? 1 polypeptide

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• DNA is template for codes
• Not direct template
• DNA transcrd ? mRNA
• mRNA direct template for polypeptides
• Translation cell uses mRNA to construct
  polypeptides

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The Genetic Code

• Discovered in 1960s
• Same code for almost all proks, euks
• Codon 3 nucleotide bases of mRNA code for 1 aa
  
• REMEMBER this info originally held as
  deoxynucleotides w/in DNA gene

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• AUG initiation codon
• Codes for met
• UAA, UAG, UGA stop codons
• Code for polypeptide synth terminn
• No spacing between codons
• Reading frame 3 nucleotides
• If insertion or deletion ? frame shift
• May ? improper aas incd into polypeptide

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• Most aas have gt1 codon
• Degenerate code
• Only aas w/ single 3-base codon met, trp
• Wobble _at_ 3rd nucleotide
• XYAG or XYUC

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• tRNAs base pair w/ mRNA codons at anticodon
• First base of mRNA codon (5?3) pairs w/ 3rd
  base of anticodon
• First two bases of mRNA codon confer most coding
  specificity

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Ribonucleic Acids

• Messenger RNA (mRNA) transcrn
• Size varies according to gene size
• So polypeptide size
• Contains genetic code w/ 3 nucleotides/aa
• Ribosomal RNA (rRNA) Helps make up ribosome
• Transfer RNA (tRNA) carries proper aa to proper
  site on ribosome

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Steps in Protein Synthesis

• Actn aas at carboxyl grp
• Aa ATP ? Aa-AMP
• Aa attd to correct tRNA
• Aminoacyl tRNA synthetase catalyzes
• Occurs in cytosol

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• Initiation
• mRNA rRNAs joined at proper sites
• Aminoacyl-tRNA enters
• Initiation factors
• Elongation
• Addition successive aas according to mRNA codons

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• Termination/Release
• Signaled by termination codon
• Release factors
• Folding/Processing of New Protein
• Proper conformation
• Removal aas addn functl grps proteolytic
  cleavage attach prosthetic grps

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The Ribosome

• Complex of enzs, rRNAs, aas to accomplish
  translation
• Made up of subunits
• Different sizes rRNA, prots, subunits
• Bacterial 70S 50S 30S subunits
• Eukaryotic 80S 60S 40S subunits
• S Svedberg unit size unit based on
  centrifugation properties

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• rRNAs have complicated 2o structures
• Needed to position mRNA/tRNA
• May act like enzymes assist in catalyzing
  formn of peptide bonds

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• Interacts w/ both m and rRNAs and aas
• At least 1 tRNA for each aa
• 1 tRNA region becomes covalently linked to its aa
• Amino acid arm

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• Covalent binding of aa to tRNA by tRNA synthetase
• 1 synthetase enz for each diff tRNA
• High-energy intermediate between tRNA aa
• Anhydride link w/ AMP
• Activated
• This intermediate links to aa arm
• Forms high energy ester linkage
• Energy held in high potential energy bonds used
  in peptide bond formn

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• tRNA synthetase has proofreading ability
• Proper aa knows to be attd to correct tRNA
• 1 region tRNA contains anticodon
• Anticodon arm
• Opposite amino acid arm
• Has 3 nucleotide base seq which interacts
  (base-pairs) w/ codon of mRNA

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• Other arms differ slightly in shape, nucleotides
• Help synthetases distinguish proper tRNAs
• Also has sites for attachment to 70 or 80S rRNA
  and mRNA

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Ingredients for Translation

• mRNA
• Has genetic code for proper aa sequence to synth
  polypeptide
• tRNA attached to aa (through high energy bond)
• Its anticodon complexes w/ mRNA _at_ codon for aa
• First base of mRNA codon base-pairs w/ 3rd base
  of anticodon on tRNA

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• rRNA assocd w/ proteins ? ribosome
• Ribosome draws all structures together properly
  to facilitate translation

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Initiation of Translation

• Begins _at_ amino terminus of new polypeptide
• New polypeptide always begins w/ met
• BUT altered met N-formylmethionine

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• Partic codon specifies fMet
• AUG
• Initiation codon
• 30S ribosomal subunit binds IF-3 and IF-1
  (proteins)
• IF-3 (or 1) Initiation Factor 3 (or 1)
• IF-3 prevents early binding of ribosomal subunits
• IF-1 prevents improper binding of tRNA to wrong
  site

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• mRNA binds 30S-IF3-IF1 complex
• So initiation codon of mRNA is at specific site
  on subunit
• Called P site (for Peptidyl site)
• Lies next to A site (for Aminoacyl site)

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• Shine-Dalgarno seq helps w/ proper placement of
  mRNA into P site
• 4-9 pur seq along mRNA _at_ 8-13 bp to 5 side of
  intitiation codon
• Recognized by rRNA of 30S ribosome
• Helps line up mRNA initiation codon _at_ P site on
  30S subunit

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• tRNA enters the structure
• Must be tRNA that carries fMet
• tRNA-fMet complex must be assocd w/ IF-2
• IF-2 must be bound to GTP
• Get 30S-IF3-IF1 complexed with mRNA (w/
  initiation codon _at_ P site)
• Anticodon region of tRNA-fMet-IF2-GTP base-paired
  to initiation codon

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• 50S subunit enters ? completed initiation complex
• GTP cleaved ? GDP Pi
• IF-1, -2, -3 disassociate
• Now have 70S ribosome
• Now P and A sites have completed conformns
• Now 3rd site (E or exit site) is formed next to P
  site

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• 3 recognition sites hold the ribosome together
• Shine-Dalgarno seq holds ribosome to mRNA
• Codon-anticodon holds mRNA to tRNA
• P site conformn holds ribosome to tRNA

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Elongation

• Ribosomal A site is empty
• mRNA w/ codon for next aa _at_ A site
• tRNA bound to aa, w/ anticodon matching codon for
  next aa is prepared
• Binds EF-Tu (a protein), which is bound to GTP
• ? Complex tRNA-aa-EF Tu-GTP
• Complex approaches empty A site on 70S ribosome
• Placed by base-pairing of codon/anticodon

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• EF Tu-GTP cleaved from tRNA and GTP hydrolyzed ?
  EF Tu-GDP Pi
• Allows time for proofreading of codon/ anticodon
  base pairing
• GDP cleaved
• EF Tu-GTP regenerated
• ? 1st aa (fMet) and aa2 in P and A sites,
  respectively
• Now in proper position to peptide bond

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• a-NH2 of aa2 attacks carbonyl of fMet
• ? aa2 tRNA now has dipeptide attached
• ? 1st tRNA now empty (uncharged w/ aa)
• REMEMBER aa had been activated by ester bond
  formn w/ tRNA
• rRNA of 50S subunit may participate in peptide
  bonding by catalyzing rxn

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• Ribosome moves 5 ? 3 along mRNA by distance of
  1 codon
• Translocation
• Due to conforml change of ribosome
• Requires EF-G attd to GTP
• EF-G translocase
• GTP cleaved from translocase and
• GTP hydrolyzed ? GDP Pi

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• GTPase impt to rate, accuracy
• EF-Tu-GTP and EF-Tu-GDP exist milliseconds
• Time for codon-anticodon pairing proofreading
• Incorrect aa-tRNAs dissoc from A site now if nec
• Note which aa attd to tRNA not checked on
  ribosome

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• ? Empty tRNA _at_ E site, tRNA-aa2-fMet _at_ P site, A
  site empty
• mRNA w/ codon for next aa _at_ A site
• Empty tRNA expelled from ribosome
• Next tRNA now positions next aa for formn of
  another peptide bond
• Note polypeptide is always attached to tRNA of
  last aa added _at_ ribosome

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Termination

• Signaled by 1 of 3 stop codons (UAA, UAG, UGA) on
  mRNA
• When stop codon is _at_ A site
• No tRNA recognizes stop codons
• Codons ARE recognized by Release Factors (RF1,
  RF2, or RF3) proteins
• One binds mRNA _at_ stop codon
• RF3 may stim release of polypeptide

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• tRNA w/ polypeptide chain attd now stalled _at_ P
  site
• Ester bond between tRNA and peptide hydrolyzed
• Also interactions between tRNA and ribosome
  weaken
• Also interactions between 30S, 50S subunits
  weaken
• Ribosome dissociates

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Polysomes

• gt1 Ribosome at a time translates a single mRNA
• Efficient use of single mRNA strand
• In bacteria, mRNA transcript translated before
  transcrn complete
• REMEMBER no nucleus, so repln, transcrn,
  transln all occur in cytoplasm
• Not true of eukaryotes

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New Proteins Are Processed

• Must be folded, altered molecularly ?
  biologically active
• REMEMBER folding, non-covalent interactions
  among 1o structure aas/functl grps ? proper 2o,
  3o, 4o structures ? functional protein

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Post-Translational Modifications

• Terminal aas modified
• fMet cleaved or formyl grp cleaved from amino
  terminus
• Terminal aas used for signaling cleaved
• Individual aas covalently modified
• Phosphn
• Carboxyl grps added
• Methyl grps added

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• Carbohydrates, isoprenes added
• ? glycoproteins, lipoproteins
• Prosthetic grps added
• Ex heme added to hemoglobin, cytochromes
• Proteolytic processing
• Polypeptide chains cleaved ? functl proteins

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Eukaryotic Translation

• REMEMBER DNA more complicated, mRNA processed
• One processing step 5 cap, polyA tail
• Impt to complexing mRNA to ribosome
• More (9) initiation factors
• Various roles, some impt to processed mRNA

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