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.

Preinitiation:

tRNA charging;

Dissociation of ribosome

Structure of tRNA

D

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

tRNA are processed from longer precursors

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.

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)

tRNA charging

Activated amino acid

At least 20 synthetases exist, one for each amino acid. Isoaccepting tRNAs are recognized by the same synthetase.

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.

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).

tRNA Synthetase

Location of varies

Each class contains about 10 enzymes

Binding of tRNA synthetase with tRNA

Class I Class II

Recognition of correct tRNA by tRNA synthetase is achieved by two steps:

AssociationAminoacylation

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.

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

Accuracy of charging tRNAIle by its synthetase depends on error control by two steps

Meaning of tRNA is determined by its anticodon alone

Structure of Ribosome

r-

70S

50S

30S

Remove Mg 2+

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.

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.

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.

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.

Recycle of ribosome and initiation factors

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

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

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

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

Removal of formyl group

Removal of methionine

Deformylase

Aminopeptidase

In bacteria and mitochondria, formyl group, and sometimes the methionine, is removed during translation.