Amino acyl t-RNA

Let us look more cl osely at the molecular events at the ribosome, where the recognition and formation of peptide bonds actually occurs. We have seen that the ribosome is composed of two different subunits, but only one of these subunits, the smaller of the two, is essential for initiation of protein synthesis, although it must be associated with the larger unit before chain elongation can proceed. Initiation also requires the presence of an energy source (supplied not by ATP but GTP), a particular amino acyl t-RNA whose anti-codon corresponds to the start here codon on m-RNA and, at least in bacteria, three soluble protein initiation factors called IF1, IF2, IF3. The ribosome has two sites for t-RNA binding, the P site and the A site, but only initiator t-RNA can bind to the P site - all other incoming amino acyl t-RNAs bind to the A site. 

Although we have said that translation of the codons could continue to the end of the m-RNA this does not usually happen. Instead translation is terminated by codons that do not bind to amino acyl t-RNA that is, they do not correspond to a particular amino acid, the full-stop codons referred to above. Release of the polypeptide is probably also mediated by a peptidyl transferase, but one which uses water rather than another amino acid as the peptidyl acceptor. GTP and yet another protein factor are also required for release, After chain release m-RNA and t-RNA remain bound to the ribosomes, but we know that the ribosomes can be used several times, so there must be an as yet undiscovered mechanism for releasing the RNA and regenerating free ribosomes. 

The dependency of bacterial RNA synthesis on protein synthesis which has been called "stringent control" has been demonstrated to take place under conditions such as aminoacid-starvation, nitrogen starvation, phosphate and Mg" starvation, energy deprivation and in strains mutated in amino-acyl-t-RNA synthetases. In all these conditions the synthesis of r-RNA and t-RNA is inhibited as far as the m-RNAs are concerned, some m-RNAs are inhibited, whereas the synthesis of others is enhanced" ". ... 

Let us consider the fact that during aminoacid starvation t-RNAs in their non amino acylated forms prevail upon the acylated t-RNAs, since protein synthesis has been driven to consume the latter. In this condition, when inhibitors of protein synthesis, whose target is in the biosynthetic steps after the formation of m-RNA-80 S ribosome complex, are added to the starved cells they prevent residual translation promoting a trickle-charging at the ribosome level that results in a sparing of charged t-RNA molecules. 

Aminoacyl adenylates (296), which are formed from protein amino acids and ATP, act as acylating agents towards t-RNAs, acylating their terminal 3 -hydroxy groups. These charged tRNAs are then used in protein synthesis. Little is known about the reactivity of aminoacyl adenylates (296), and studies are now reported of a model compound, alanyl ethyl phosphate (297). As expected, hydrolysis in both acid and base involves attack at the C=0 group of (297) with departure of ethyl phosphate. Metal ions (Cu +, Zn +) were found to act as catalysts of the hydrolysis. 

Amino acyl 5 -adenylates have been known as products in the first stage of activation for the amino acids in protein biosynthesis before they are coupled to t-RNA. The... 

The reason for the apparent requirement for amino acids in the synthesis of RNA in vivo has not so far received a clear-cut answer from our knowledge of the in vitro system. In the adenylates we have a potential point of contact between the two processes since, as we have seen, both the amino acyl and the adenylyl groups are potentially activated, and hence are potential precursors for protein and for RNA syntheses, respectively. In actual practice, however, we can have either a C—O or a P—O split, but not both at the same time 1 mole of adenylate could at most yield one-half mole of peptide, and one-half mole of polynucleotide. This makes it quite unlikely that the adenylates are a common precursor, i.e., give rise to the simultaneous synthesis of protein and of RNA, as was thoi t by some people when these substances first made their appearance. Such a mode of RNA synthesis would further require that, in addition to the adenylates, there be mixed anhydrides of amino acids with the other three nucleotides, and, so far, no evidence of such compounds has been found. 

Inq)roved routes have been develed for the synthesis of pdCpA. acylated at 0-3 of dte adenosine unit with non-natural aminoacids. The resultant aminoacyl compounds can be inctxporated into t-RNA s by enzymic ligation, and the novel amino add can then be inserted into aiwotein.231... 

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