Initiation of Translation in Bacteria Chapter 17

Initiation of Translation in Bacteria Two important events- before translation initiation - Generate a supply of aminoacyl-tRNAs Amino acids must be covalently bound to tRNAs - tRNA charging - Dissociation of ribosomes into their two subunits The cell assembles the initiation complex on the small ribosomal subunit The two subunits must separate to make assembly possible Two important events must occur before translation initiation can take place - Process of bonding tRNA to amino acid is called tRNA charging

tRNA Charging All tRNAs have same 3 bases at 3’-end (CCA) Terminal adenosine is the target for charging with amino acid Amino acid attached by ester bond between Its carboxyl group 2’- or 3’-hydroxyl group of terminal adenosine of tRNA

Aminoacyl-tRNA Synthetase Activity Aminoacyl-tRNA synthetases join amino acids to their cognate tRNAs - done in a two-step reaction: Begins with activation of the amino acid with AMP derived from ATP The energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA

Dissociation of Ribosomes E. coli ribosomes dissociate into subunits at the end of each round of translation IF1 actively promotes this dissociation IF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome 70S ribosomes of E.coli contain one 30S and one 50S subunit. Each subunit has one or two ribosomal RNAs and large collection of ribosomal proteins. The 30S subunit binds the mRNA and the anticodons ends of tRNAs. It is the decoding agent of ribosome that reads the genetic code in mRNA and allows binding with appropriate aminoacyl-tRNAs. The 50S subunit binds the ends of tRNAs that are charged with amino acids and has peptidyl transferase activity that links amino acids together through peptide bonds.

Formation of the 30S Initiation Complex When ribosomes - dissociated into 50S and 30S subunits - cell builds a complex on the 30S subunit: mRNA Aminoacyl-tRNA Initiation factors IF3 binds by itself to 30S subunit IF1 and IF2 stabilize this binding IF2 can bind alone - but is stabilized with help of IF1 and IF3 When ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit. IF1 does not bind alone

First Codon and the First Aminoacyl-tRNA Prokaryotic initiation codon is: Usually AUG, can be GUG, rarely UUG Initiating aminoacyl-tRNA is N-formyl-methionyl-tRNA N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide This amino acid is frequently removed from the protein during maturation The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNA

Binding mRNA to the 30S Ribosomal Subunit Binding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing between Short RNA sequence Shine-Dalgarno sequence Upstream of initiation codon Complementary sequence 3’-end of 16S rRNA

Initiation Factors and 30S Subunit Binding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3 - Assisted by IF1 and IF2 All 3 initiation factors have bound to the 30S subunit at this time IF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complex GTP is also required for IF2 binding at physiological IF2 concentrations The GTP is not hydrolyzed in the process

Complete 30S Initiation Complex The complete 30S initiation complex contains one each: 30S ribosomal subunit mRNA fMet-tRNA GTP Factors IF1, IF2, IF3

Formation of the 70S Initiation Complex GTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complex This GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunit Hydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin

Bacterial Translation Initiation IF1 influences dissociation of 70S ribosome to 50S and 30S Binding IF3 to 30S, prevents subunit reassociation IF1, IF2, GTP bind alongside IF3 Binding mRNA to fMet-tRNA forming 30S initiation complex Can bind in either order IF2 sponsors fMet-tRNA IF3 sponsors mRNA Binding of 50S with loss of IF1 and IF3 IF2 dissociation and GTP hydrolysis

Initiation in Eukaryotes Eukaryotic Begins with methionine No Shine-Dalgarno mRNA have caps at 5’end Bacterial N-formyl-methionine Shine-Dalgarno sequence to show ribosomes where to start

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