Aminoacyl-tRNA codon-specific binding

Codon-specific binding of an aminoacyl-tRNA (decoding)... 

Codon-specific binding of an aminoacyl-tRNA (decoding). The binding of an aminoacyl-tRNA to the A site of the 70S or 80S initiation complex depends upon a protein called elongation factor Tu (EF-Tu... 

The answer is b. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) During the course of protein synthesis on a ribosome, peptidyl transferase catalyzes the formation of peptide bonds. However, when a stop codon such as UAA, UGA, or UAG is reached, aminoacyl-tRNA does not bind to the A site of a ribosome. One of the proteins, known as a release factor, binds to the specific trinucleotide sequence present. This binding of the release factor activates peptidyl transferase to hydrolyze the bond between the polypeptide and the tRNA occupying the P site. Thus, instead of forming a peptide bond, peptidyl transferase catalyzes the hydrolytic step that leads to the release of newly synthesized proteins. Following release of the polypeptide, the ribosome dissociates into its major subunits. 

Codon-specific binding of an aminoacyl-tRNA (decoding). The binding of an aminoacyl-tRNA to the A site of the 70S or SOS initiation complex depends upon a protein called elongation factor Tu (EF-Tu or elFl in eukaryotes), which is present as a mixed dimer with a second protein, EF-Ts. In E. coU EF-Ts is a stable 35-kDa protein, while Tu is a 43-kDa soluble protein present in a large excess over Ts. Tu is one of the most abimdant soluble proteins in bacterial cells and accoimts for about 5% of the total protein. Most of the tRNAs in a bacterial cell are present as complexes with Tu. Tu may also have fimctions other than in protein s5mthesis and is foimd associated with the plasma membrane as well as with ribosomes. 

For codon-specific binding of aminoacyl-tRNA to the ribosome the tRNA must form a complex with the elongation factor Tu. The complex formation manifests itself also in the imino resonance region of the NMR spectrum of the tRNA. Since the molecular mass of tRNA (25,000 Da) is only about one third of the molecular mass of the aminoacyl-tRNA-EF-Tu GTP complex (70,000 Da) the complex formation should become apparent by an increase of the line widths of the imino resonances. This was indeed observed with a complex of yeast Phe-tRNA and EF-Tu GTP from T. thermophilus (Fig. 19.14). 

Most transfer RNAs have common parts and uncommon parts. The common parts facilitate binding of the aminoacyl-tRNAs to common sites on the ribosome. The uncommon sites permit specific reactions with charging enzymes that covalently attach the correct amino acids to the correct tRNA. Another uncommon site on the tRNAs is the anticodon, which leads to specific complex formation with the complementary codon site on the messenger. 

Obviously, each codon present within mRNA must correspond to a specific amino acid. Nirenberg found that trinucleotides of known base sequence could bind to ribosomes and induce the binding of specific aminoacyl-tRNAs (i.e., tRNAs with amino acids covalently attached). As a result of these and the earlier experiments, the relationship between all 64 codons and the amino acids they specify (the entire genetic code) was determined by the mid-1960s (Table 15.1). 

Further study of the specificity of aminoacyl-tRNA binding of EF-Tu resulted in the solution of the following problem the chain initiator in E. coli is fMet-tRNA( which is synthesized by formylation of Met-tRNAj. There is a different Met-4RNA species, Met-tRNA which provides methionyl residues for internal positions of the peptide chain. Furthermore as initiator codons in E. coli, GUG, as well as AUG, spedfy fMet-tRNAt, whereas as codons for internal aminoacyl residues GUG stands for Val-tRNA and AUG for Met-tRNA . 

Addition of each incoming amino acid requires the cooperation of three elongation factors. Elongation factor Tu (EF-Tu) is the most abundant protein in E. coli, with about 100,000 copies per cell, or 5% of the cell s protein. This protein is a GTPase, and the EF-Tu GTP complex specifically binds aminoacyl-tRNAs (AA-tRNAs). Formation of the ternary complex (EF-Tu GTP AA-tRNA) protects the ester bond (linking the amino acid to its cognate tRNA) from hydrolysis, and transports the AA-tRNA to the ribosomal A-site. Once the correct codon-anticodon interaction is confirmed, ribosome-triggered hydrolysis of... 

Initiation. Translation begins with initiation, when the small ribosomal subunit binds an mRNA. The anticodon of a specific tRNA, referred to as an initiator tRNA, then base pairs with the initiation codon AUG. Initiation ends as the large ribosomal subunit combines with the small subunit. There are two sites on the complete ribosome for codon-anticodon interactions the P (peptidyl) site (now occupied by the enitiator tRNA) and the A (aminoacyl) site. In both prokaryotes and eukaryotes, mRNAs are read simultaneously by numerous ribosomes. An mRNA with several ribosomes bound to it is referred to as a polysome. In actively growing prokaryotes, for example, the ribosomes attached to an mRNA molecule may be separated from each other by as few as 80 nucleotides. 

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