Translation

1
Translation

• Chapter 27

2
Central Dogma

• Genetic code contained w/in 4 deoxy-nucleotide
  bases of DNA
• 1 gene ? 1 polypeptide
• DNA is template for codes, but not direct
  template
• DNA transcrd ? mRNA
• mRNA is direct template for polypeptides
• Translation cell uses mRNA to construct
  polypeptides

3
The Genetic Code

• Discovered in 1960s (27-7)
• Same code for almost all prokaryotes, eukaryotes
• Codon 3 nucleotide bases of mRNA that code for
  1 aa
• REMEMBER this info was originally held as
  deoxynucleotides w/in gene of DNA
• Most aas have gt1 codon
• Only aas w/ single 3-base codon met, trp
• Wobble _at_ 3rd nucleotide

4
Fig.27-7
5
The Genetic Code contd

• AUG initiation codon
• Codes for met
• UAA, UAG, UGA stop codons
• Code for termination of polypeptide synth
• No spacing between codons
• Reading frame 3 nucleotides
• If insertion or deletion ? frame shift (27-3)
• May ? improper aas incd into polypeptide

6
Ribonucleic Acids

• Messenger RNA (mRNA) described in transcrn
  (Chpt. 26)
• Size varies according to gene size
• So polypeptide size
• Contains genetic code w/ 3 nucleotides/aa
• Ribosomal RNA (rRNA) RNA that helps make up the
  ribosome
• Transfer RNA (tRNA) carries aa to proper site
  on ribosome

7
The Ribosome

• Complex of enzs, rRNAs, aas to accomplish
  translation
• Made up of subunits
• Different sizes rRNA, prots, subunits (27-11)
• Bacterial 70S 50S 30S subunits
• Eukaryotic 80S 60S 40S subunits
• S Svedberg unit size unit based on
  centrifugation properties

8
Fig.27-11
9
Fig.27-11
10
The Ribosome contd

• rRNAs have complicated 2o structures (27-12)
• Needed to position mRNA/tRNA
• May act like enzymes assist in catalyzing
  formn of peptide bonds

11
Transfer RNA (27-15)

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

12
Transfer RNA contd

• Covalent binding of aa to tRNA by tRNA synthetase
• 1 synthetase enz for each diff tRNA
• Forms high-energy intermediate between tRNA aa
• Anhydride link w/ AMP (27-16)
• Activated
• This intermediate links to aa arm
• Forms high energy ester linkage
• Energy held in these high potential energy bonds
  is used in peptide bond formn

13
Fig.27-16
14
Fig.27-16
15
Transfer RNA contd

• tRNA synthetase has proofreading ability
• Where proper aa knows to be attd to correct
  tRNA
• 1 region of tRNA contains anticodon
• Anticodon arm
• Opposite Amino acid arm
• Has seq of 3 nucleotide bases which interact
  (base-pair) w/ codon of mRNA (27-14)

16
Fig.27-14
17
Transfer RNA contd

• Other arms differ slightly in shape, nucleotides
• Help synthetases distinguish proper tRNAs (27-18)
• Also has sites for attachment to 70 or 80S rRNA
  and mRNA

18
Ingredients for Translation

• mRNA
• Contains genetic code for proper aa sequence to
  synthesize polypeptide
• tRNA attached to aa (through high energy
  bond)(27-17)
• This complexes w/ mRNA _at_ codon for aa
• First base of mRNA codon base-pairs w/ 3rd base
  of anticodon on tRNA (27-8)

19
Fig.27-17
20
Fig.27-8
21
Ingredients for Translation contd

• rRNA assocd w/ proteins ? ribosome
• Ribosome draws all structures together properly
  to facilitate translation
• Note translation has initiation, elongation,
  termination steps. Book uses E. coli system as
  model

22
Initiation of Translation

• Begins _at_ amino terminus of new polypeptide
• New polypeptide always begins w/ met
• BUT altered met N-formylmethionine (p.1044)
• fMet has particular codon that specifies it
• AUG
• Called initiation codon

23
Initiation contd

• 30S subunit of ribosome binds IF-3 and IF-1
  (27-22)
• IF-3 (or 1) Initiation Factor 3 (or 1)
  (proteins) (Table 27-9)
• IF-3 prevents early binding of ribosomal subunits
• IF-1 prevents improper binds of tRNA to wrong
  site
• mRNA binds 30S-IF3-IF1 complex
• Binding such that initiation codon of mRNA is at
  specific site on subunit
• Called P site (for Peptidyl site)
• Lies next to A site (for Aminoacyl site)

24
Fig.27-22
25
Initiation contd

• Shine-Dalgarno sequence helps w/ proper placement
  of mRNA into P site (27-23)
• Specific seq along mRNA _at_ partic site relative to
  intitiation codon
• Recognized by rRNA of 30S ribosome
• Helps line up mRNA initiation codon _at_ P site on
  30S subunit

26
Fig.27-23
27
Initiation contd

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

28
Fig.27-22
29
Initiation contd

• 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

30
Fig.27-22
31
Initiation contd

• 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

32
Elongation contd

• A site on ribosome is empty
• mRNA w/ codon for next aa is _at_ A site
• tRNA bound to aa, w/ anticodon that matches codon
  for next aa is prepared (27-25)
• Binds EF-Tu (Elongation Factor Tu, a protein),
  which is bound to GTP
• ? Complex of tRNA-aa-EF Tu-GTP
• Complex approaches empty A site on 70S ribosome
• Placed by base-pairing of codon/anticodon

33
Fig.25-25
34
Elongation contd

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

35
Elongation contd

• a-NH2 of aa2 attacks carbonyl of fMet (27-26)
• ? 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

36
Fig.27-26
37
Elongation contd

• Ribosome moves 5 ? 3 along mRNA by distance of
  1 codon (27-27)
• Called translocation
• Due to conforml change of ribosome
• Requires EF-G attd to GTP
• EF-G translocase
• GTP cleaved from translocase and
• Also, GTP hydrolyzed ? GDP Pi

38
Fig.27-27
39
Elongation contd

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

40
Termination of Translation

• Signaled by 1 of 3 stop codons (UAA, UAG, UGA) on
  mRNA (27-28)
• When stop codon is _at_ A site
• No tRNA recognizes these codons
• These codons ARE recognized by Release Factors
  (RF1, RF2, or RF3) proteins
• One of these binds mRNA _at_ stop codon
• RF3 may stimulate release of polypeptide

41
Termination contd

• 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

42
Fig.27-28
43
Polysomes

• gt1 Ribosome at a time translates a single mRNA
  (27-29)
• Efficient use of single mRNA strand
• In bacteria, mRNA transcript is translated before
  transcrn complete (27-30)
• REMEMBER no nucleus in prokaryotes, so repln,
  transcrn, transln all occur in cytoplasm
• Not true of eukaryotes

44
Fig.27-29
45
Fig.27-30
46
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
• Post-translational modifications
• Terminal aas modified
• fMet cleaved or its formyl grp cleaved from amino
  terminus
• Terminal aas used for signaling cleaved

47
Post-translational modifications

• Individual aas covalently modified
• Phosphn

48
Post-translational modifications

• Individual aas covalently modified
• Carboxyl grps added
• Methyl grps added

49
Post-Translational Modifications

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

50
Eukaryotic Translation

• REMEMBER DNA more complicated, mRNA processed
• One processing step 5 cap, polyA tail
• Play a role in complexing of mRNA to ribosome
  (27-24)
• More (9) initiation factors (Table 27-9)
• Various roles, some impt to processed mRNA

51
Fig.27-24