Prokaryotic Translation
description
Transcript of Prokaryotic Translation
![Page 1: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/1.jpg)
Prokaryotic TranslationThree stages
Initiation: binding of ribosome (containing rRNAs and proteins) and aminoacyl tRNA to mRNA.
Elongation: addition of one aa at a time to the growing polypeptide chain.
Termination: release of finished polypeptide from tRNA and dissociation of ribosome from mRNA.
![Page 2: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/2.jpg)
Preinitiation:
tRNA charging;
Dissociation of ribosome
![Page 3: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/3.jpg)
Structure of tRNA
D
![Page 4: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/4.jpg)
Structure of aminoacyl-tRNA
ACC I
Length: 76 (74-95) residues.
Extra arm (variable loop):
Class I tRNA: 3-5 bases
Class II tRNA: 13-21 bases; ~5 bases in the stem.
Additional types of base pairing:
G·U, G·, A·.
Less stable
Positions:
Invariant: maintain 2O structure
Semiinvariant (other Pu or Py)
Name of tRNA:
tRNA1 tRNA2
Name of charged tRNA: Tyr-tRNA
Tyr Tyr
![Page 5: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/5.jpg)
tRNA are processed from longer precursors
![Page 6: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/6.jpg)
Modified nucleosides found in tRNA
The bases are
modified (>50
different types)
after transcription
by specific tRNA-
modifying
enzymes to affect
the efficiency of
charging and
pairing properties.
![Page 7: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/7.jpg)
3-D Structure of tRNA
Tertiary structure of tRNA is created by H-bonding:
Secondary H-bonds;
Tertiary H-bonds (formed between unpaired invariant and semiinvariant bases)
RNA-RNA double helices (11 bp/turn)
![Page 8: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/8.jpg)
tRNA charging
Activated amino acid
At least 20 synthetases exist, one for each amino acid. Isoaccepting tRNAs are recognized by the same synthetase.
![Page 9: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/9.jpg)
tRNA charging
Recognition depends on an interaction between a few points of contact in tRNA, mostly at the acceptor stem and anticodon, and a few amino acids constituting the active site in the enzyme.
![Page 10: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/10.jpg)
Binding of tRNA synthetase with tRNATwo classes of tRNA synthetase:
Class I tRNA synthetases
Aminoacylate the 2’-OH group of the terminal A of the tRNA.
Approach the tRNA from the D-loop and acceptor stem minor groove side.
Class II tRNA synthetases
Aminoacylate the 3’-OH group of the terminal A of the tRNA.
Approach the tRNA from the variable arm and acceptor stem major groove side (the opposite side of tRNA that contacts the class I enzyme).
![Page 11: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/11.jpg)
tRNA Synthetase
Location of varies
Each class contains about 10 enzymes
![Page 12: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/12.jpg)
Binding of tRNA synthetase with tRNA
Class I Class II
![Page 13: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/13.jpg)
Recognition of correct tRNA by tRNA synthetase is achieved by two steps:
AssociationAminoacylation
![Page 14: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/14.jpg)
Recognition of correct
amino acid by tRNA sy
nthetase is also achie
ved by two steps (ever
y synthetase undergo
es proofreading at eith
er stage), which occur
s only in the presence
of cognate tRNA.
![Page 15: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/15.jpg)
Ile-tRNA synthetase has two active sites for sieving the cognate amino acid
Synthetic site: activation of amino acid
Editing site: hydrolysis of incorrrect aminoacyl-tRNA
![Page 16: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/16.jpg)
Accuracy of charging tRNAIle by its synthetase depends on error control by two steps
![Page 17: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/17.jpg)
Meaning of tRNA is determined by its anticodon alone
![Page 18: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/18.jpg)
Structure of Ribosome
r-
![Page 19: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/19.jpg)
70S
50S
30S
Remove Mg 2+
![Page 20: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/20.jpg)
Arrangement of proteins and 16S rRNA in S30 subunit
Central domainCentral domain
Both 30S and 50S subunits are self-assembled in vitro. In 30S subunit, S4 and S8 bind to 16S rRNA first, other proteins then join sequentially and cooperatively.
RNA is concentrated at the interface with the 50S subunit.
![Page 21: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/21.jpg)
16S rRNA
Secondary structure of 16S rRNA and interaction of this RNA with proteins and tRNA were studied by primer extension and crosslinking, and other techniques.
![Page 22: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/22.jpg)
Features of rRNAsrRNAs have considerable 2o structure (This is analyzed by comparing the sequences of corresponding rRNAs in related organisms).
About 2% of the residues in rRNAs are methylated, which may be important for ribosomal function.
Interaction of rRNA with some ribosomal protein induce conformational change of rRNA so that it can interact with another protein.
rRNA interacts with mRNA or tRNA at each stage of translation. The proteins are necessary only to maintain the rRNA in a structure in which rRNA can perform the catalytic function. Conformation of rRNAs is flexible during protein synthesis.
The 3’ terminus of 16S rRNA pairs with the SD sequence of mRNA at initiation.
rRNA contacts the tRNA at parts of the structure that are universally conserved.
![Page 23: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/23.jpg)
Translation initiation
1. Dissociation of ribosome.
2. Binding of IF-3 to 30S subunit to prevent reassociation of ribosome.
3. Binding of IF-1 and IF-2 (with GTP) alongside IF-3.
4. Binding of mRNA and fMet-tRNAfMet t
o form 30S initiation complex.
5. Binding of 50S subunit with loss of IF-1 and IF-3.
6. Dissociation of IF-2 with hydrolysis of GTP to form 70S initiation complex.
![Page 24: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/24.jpg)
Recycle of ribosome and initiation factors
![Page 25: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/25.jpg)
Ribosomes bind to mRNA at a special sequence
Ribosomal protein
S12 and 16S rRNA,
are responsible for
recognition of the
SD sequence. A
conserved sequence
near the 3’-end of
16S rRNA pairs with
the SD sequence.
Ribosomal binding to the SD sequence provides a means for controlling gene expression.
IF-3 is the primary factor for mRNA binding to ribosome. IF-1 and IF-2, which bind near IF-3, assist assembly of 30S initiation complex.
(about 30 nt)
mRNA sequence protected by ribosome
![Page 26: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/26.jpg)
This initiator tRNA recognizes codons AUG (90%), GUG (8%), or UUG (1%) that lies within a ribosome-binding site.
fMet-tRNAf as initiator tRNA
Formation of fMet-tRNAf
tRNAf is different fro
m tRNAm ; the former
goes to the first AUG codon and the methionine of the later can not be formylated.
Met
Met
![Page 27: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/27.jpg)
Formylation is not strictly necessary for the initiator tRNA to function in initiation.
It is the tRNA part of fMet-tRNAf that makes it the
initiating aminoacyl-tRNA.
IF-2 ensures only the initiator tRNA goes to the P-site at initiation.
Features of fMet-tRNAf
Met
![Page 28: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/28.jpg)
IF2-GTP joins complex
Initiator tRNA joins
30S-mRNA complex
50S joins and all factors are released
IF2 is needed to bind fmet-tRNAf to 30S-mRNA complex
Formation of 70S initiation complex
![Page 29: Prokaryotic Translation](https://reader033.fdocuments.net/reader033/viewer/2022051217/56814de5550346895dbb558b/html5/thumbnails/29.jpg)
Removal of formyl group
Removal of methionine
Deformylase
Aminopeptidase
In bacteria and mitochondria, formyl group, and sometimes the methionine, is removed during translation.