Protein Synthesis and modification

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???? ??????????? Protein Synthesis and
modification
??? ??
???????????????? ???
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? ?
?????mRNA?,mRNA???????????????????????????
??
1?  ?? 20????
2?  ?? ATP?GTP
3?  ??? ????????????(Mg,K )
4?  ????tRNA
5?  ??mRNA(??A.A???????)
6?  ??????(rRNA????)
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??? ???????
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??? ???????????
???? mRNA????5?3???,???????????,?????????(
codon)?
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(No Transcript)
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???????64????,??61 ??????????AUG ??????
?Met?????
????????????UAA?UAG?UGA,
?????????tRNA??,??? ?????????
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?????????(5??)
1???????? 2???????? 3????????? 4???????? 5????????
6??????
??
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1????????
????????????????????????(degeneracy)?
???????????????????????(synonymous codon)
?????????
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2????????
?????? ???????????(reading frame)
??????????,?????? ????,???????????
?????????
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3?????????(non-overlapping)
???????????????????????????RNA????,????
???????????
?????????
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4???????? P423
???????????????,?????????
???,?????????????????,Crick????????????????????,?
??? wobble.
?????????
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1966?F.Crick???????(wobble hypothesis)
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(No Transcript)
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tRNAs

• Secondary Tertiary structure
• Isoacceptor tRNAs (????tRNA)
• Different tRNAs carrying the same aa
• tRNA identity- 2nd genetic code P431
• Special sequence elements on tRNAs that can
  be recognized by aaRS and then determine which aa
  is charged
• Positive elements Negative elements
• Some special tRNAs
• 1) Initiator tRNA tRNAf Met tRNAiMet
• 2) Suppressor tRNA(??tRNA)
• 3) tmRNA

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5????????
??????????? ?
?????????
??
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General features

• With mRNA as template, tRNA as carrier for aa,
  ribosome as assembly site
• Translational polarity
• 1) Extends in N-end ? C-end
• how to prove?
• 2) Reads mRNAs in 5 ? 3
• Triplet codon
• 1) How many bases determine one aa?
• three determine one
• 2) Cracking of the genetic code
• 3) Features of the genetic code
• Ribosomes recognize aa-tRNA just by virtue of the
  base-pairing interaction between codons and
  anticodons
• Wobble hypothesis

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The Nature of the Genetic Code

• All the codons have meaning 61 specify amino
  acids, and the other 3 are "nonsense" or "stop"
  codons
• The code is unambiguous - only one amino acid is
  indicated by each of the 61 codons (wobble???????
  )
• The code is degenerate - except for Trp and Met,
  each amino acid is coded by two or more codons
  (???????)
• Codons representing the same or similar amino
  acids are similar in sequence

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• 2nd base pyrimidine usually nonpolar amino acid
• 2nd base purine usually polar or charged aa
• The code is not overlapping(????????)
• The base sequence is read from a fixed starting
  point, with no punctuation(???????)
• Universal Unusual

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??? ????????
?????(ribosome)???????????
????????????????,????????????,??????????????????
??????????,??????????????
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?????(ribosome)
???????
5S rRNA, 23S rRNA
50S
34????
70S
16S rRNA
30S
21????
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???????
5SrRNA,5.8SrRNA,28SrRNA
60S
49????
80S
18SrRNA
40S
33????
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(No Transcript)
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????RNA??????? tRNA??????????
?????????????????????tRNA???
tRNA??????????????? ??????
1)  3?CCA????????? 2)   ????tRNA??????
3)   ???????,??????? ??????? 4)   ??????
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Functional sites on Ribosome

• Three sites for tRNA
• 1) The A (acceptor) site - where aa-tRNAs come
  in (except the first one) and where peptidyl-tRNA
  is after peptide bond formation and before
  translocation.
• 2) The P (peptidyl-tRNA) site - where
  peptidyl-tRNA is before peptide bond formation.
• 3) The E (exit) site - where the uncharged tRNA
  from the P site goes after translocation.
• Peptidyl transferase - the active site that
  catalyzes formation of the peptide bond (23S rRNA
  in Prokaryotes)
• Polypeptide exit channel
• mRNA binding site

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(No Transcript)
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(No Transcript)
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(No Transcript)
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????-tRNA??????????? P429 ??????-tRNA?????????
??tRNA??? , ???????? a)      ????? b)      
??tRNA(?L??????) c)       ????????
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E.Coli????????
1.?????? ????????-tRNA???
(aminoacyl-tRNA synthetase)??,?????
????
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(No Transcript)
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????-tRNA????tRNA??? ???????????????
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?????????????????
????-tRNA???(P432) ????,???????????? ???????,????
?????-tRNA????,???N-??????-tRNA??????
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?E.Coli??????????????(AUG)????tRNA???????tRNA(fMe
t-tRNAf Met),????tRNA?
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??tRNA??????????-tRNA???
???
??????
tRNA
Met-tRNA
Met
N10-??FH4
FH4
(fMet-tRNAf)
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(No Transcript)
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Structure of N-formyl-methionyl-tRNAMet
Differences with other tRNAs
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????2????Met?tRNA,????????AUG???,??????????????
(1)tRNAf Met ??Met?????,???tRNA,??????????
(2)tRNAmMet??Met??????,???????,??????????
??AUG?GUG??????,????????????,???????????,?
????????????Met????????????aa???,??AUG??Met,GUG??v
al?
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????????????? 1?30S-mRNA??????
????????3(IF3) P???????? A????????? ???????P?? S
D?? Shine-Dalgarno???AUG?? ???????AG?,?30S??16Sr
RNA????????
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mRNAs

• Prokaryotic mRNAs
• Usually polycistronic
• Eukaryotic mRNAs
• Usually monocistronic

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Shine-Dalgarno Sequences recognized by E.coli
ribosomes
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2.?????

• mRNA???????AUG,???GUG?
• ?????????10????????????????????SD??(Shine-Dalgarno
  ??),???310????,?????16srRNA 3?????????,???????mR
  NA????

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2?30S??????(IF1,IF2,GTP)
??????? IF1?????,??IF3,IF2???,???IF1???,mRNA-30S-I
F3????fMet-tRNAf,GTP???,?? ??IF3?? ??30S?????,
30S???mRNA-fMet-tRNAf?
?????
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SD sequence
inactive 70S ribosome
30S initiation complex
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GDP Pi
70S initiation complex
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3. ?????
???????????????????(elongation factor,EF)
?????EF?EF-T?EF-G??

• EF-T????T,????????????????????peptidyl
  transferase,????????????T ?

• EF-G???????????????GTP,?GTP???,????????????????,
  ????GTP,???GTP??????G,EF-G?

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• EF-T??Tu?Ts??????????,
• EF-Tss?stable???,??????,
• EF-Tu u?unstable,???????
• EF-Tu???????-tRNA????????

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???????3?

• ??-tRNA??????A?
• ?????
• ??

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(1)??-tRNA??????A?
??????????,?Ts?Tu,?? ?
?GTP?EF-Tu?GTP???-tNRA?????? ???,????A??
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(2)?????
????????(transpeptidation),??????(peptidyl
transferase)??,??????????,????????????????23s
rRNA??(????????ribozyme)??????????????????????????

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(No Transcript)
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(3)??(translocation)

• ????????
• ???tRNA??P??
• ???tRNA?A???P?

• ????mRNA?5?3??
• ???????????
• ????EF-G?GTP?

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(No Transcript)
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4.????????
????(release factor RF)?????????????????

• RF-1??UAA?UAG
• RF-2??UAA?UGA
• RF-3RF-3?GTP???
• ??,???GTP???
• ?,???????RF-1
• ?RF-2????

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(No Transcript)
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(No Transcript)
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(No Transcript)
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3?70S?????
30S???????50S????,???????????70S?????,??GTP
???GDP?Pi,IF1?IF2,??????fMet-tRNAf???????P??,
???A???????????-tRNA, ???????????
?????
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???????????? 1??? (EF-Ts,EF-Tu,GTP)
EFTu??GTP??, ????-tRNA?????????,??
70S????????,???? EFTu-GDP,EFTu-GDP??EFTs?GTP??,??
??????EFTu-GDP
??fMet-tRNAf?,????-tRNA???EFTu,GTP??????70
S????A??
?????
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2???
?50S?????????????P???A??,??????????,?50S??
??????????P????tRNA???????????tRNA,A????tRNA??????
???,??????K???
?????
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3????? (EF-G,GTP)
???? ????tRNAf?? ???- tRNA?A???P?
?mRNA??3??? ???codon??ribosome?A?
????????mRNA(53)? ?????
?????
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?????????? 1????????
????,??????(RF-1, RF-2) RF-1???UAA,
UAG RF-2???UAA, UGA
?????
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2???
????? RF-1 RF-2???A?,????????????R1,R2??????P??
????????????????,?????-tRNA???????-tRNA?,???????
?????
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3???
??tRNA?70S??????,?????????mRNA,???50S?30S??,?
???????????????,?????
?????
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???????????????

1. ??
2. ??

????????????
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??
????????????,???? ??????
?????????????????,?? 1? 
???????????? 2?  ??mRNA?????????????????????
? 3?  ????????? 15A.A/second
???????????????????.
cell??1?2??????????5A.A/second
?????(polyribosome)
?????????????
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5.?????(polyribosome, or polysome)
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??
???ATP?GTP???? ??????,GTP?ATP??,????
??????????????,??????????,??????????????????????,?
?GTP?GDP,???????????
?????????????
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??
??6?A. A oooooo, ???????ATP(???A.A????)?
?     n?A,A????2n?ATP?????? ?    
??-tRNA????n-1???A?
GTPGDPPi ?     ????,??n-1???A?
GTPGDPPi ?     ????? 1GTP ??,?????n?A.A
?????? 4n-1??????
?????????????
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(?)????????????
????
????100?aa?????????????
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?? ??????????????
??????
1?mRNA???????, ???????????,???????
2????5?????AUG?????????
3???AUG??????????????cap(??????)????5?
????????????
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(?)????????????
???????????????????,??????,????????
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1.??? ?????????,?80S,????60S?40S?????
2.??tRNA ??????????Met?tRNA,????Met?tRNA???t
RNA ,?tRNAiMet,??tRNA?? Met?,???????????????tRNA
Met?

• ?????
• ?????????AUG,?????SD??,???
• mRNA????5??AUG??????, 40S????
• mRNA 5????????,?3?????,????
• AUG???,??????ATP?

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4. ????
???????????,???????????10?,???eIF-n,e????
,?eIF-1,eIF-2,eIF-2A,eIF-3,eIF4A?4B?4C?4D,eIF-5,eI
F-6,??eIF-2???????,??????????(cap-binding
protein)??GTP???tRNA?????????,?????
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5.?????????
??????????EF-1a?EF-1ßr,?????????EF-Tu?EF-Ts
??????EF-2????????EF-G(EF-2???GTP??,??,????)?
???????????????????eRF,???3???????
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???????
????????
????????
??
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???????????
???? ??????????-tRNA3????AMP??????????????
???????????????,????????,???????,????????-????????
????????,???Protein???????
????????????
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???????????
???????,?????????? ?????????
??????70S???,??80S?????? ?????????80S???,???
?????????? ?????????????????????,??????????
????????????
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??? ?????????
???????????????????
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??? ????????