Structure and Function of Transfer Ribonucleic Acids

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

The focus of this proposal is initiator tRNAs and their role in initiation of protein synthesis. The major objectives are to investigate (a) the molecular mechanism of recognition of E. coli initiator tRNA by components of the translational machinery and (b) the function of initiator tRNA in initiation of protein synthesis directed by leaderless mRNAs in E. coli. A further objective is to investigate the relationship between the structure and function of archaeal initiator tRNAs and the translational machinery of archaea in general. An important question is the molecular mechanism of recognition of the formylmethionyl-tRNA (fMet-tRNA) by IF2. A combination of biochemical, genetic and structural approaches will be used. These include (1) crosslinking of IF2 carrying benzoyl-phenylalanine residues at specific sites to fMet-tRNA and identification of crosslinking sites, (2) isolation of suppressor mutations in IF2, which can rescue the initiation defect of mutant initiator tRNAs which are blocked in formylation of Met-tRNA to fMet-tRNA and (3) crystal structure analysis of fMet-tRNA complexed to IF2 or the C-terminal fMet-tRNA binding domain of IF2. A related question is whether IF2 acts as a carrier of fMet-tRNA to the ribosome. Work in vitro and in vivo suggests that this is the case, however, more direct experiments are necessary. Another question being addressed is (1) whether the AUG initiation codon is an absolute requirement for translation of leaderless mRNAs in E. coli and (2) whether there are any cis-acting signals in leaderless mRNAs besides the AUG initiation codon. For example, does the 5'-triphosphate group proximal to the AUG codon of leaderless mRNAs contribute in any way towards their activities? Other important aims are: (i) To identify the requirements in an initiator tRNA for translation initiation and initiator-elongator discrimination in archaea. While there is much known now about these requirements in E. coli and eukaryotic initiator tRNAs, virtually nothing is known about archaeal initiator tRNAs. It is timely and important to begin such work on archaeal initiator tRNAs and the archaeal translational machinery using the in vivo and in vitro approaches used successfully for E. coli and mammalian initiator tRNAs. (ii) To investigate genetic suppression mechanisms in archaea. In eubacteria, ochre suppressor tRNAs, which read the UAA stop codon also read the amber stop codon UAG. In eukaryotes, however, ochre suppressors are specific for UAA. Nothing is known about genetic suppression in archaea. The reporters developed for work on initiator tRNAs can also be used for these studies. (iii) To investigate the mechanism by which an archaeal isoleucine tRNA translates specifically the isoleucine codon AUA but not the other isoleucine codons AUU and AUC, or the methionine codon AUG. Eubacteria use one mechanism to specifically read AUA whereas eukaryotes use another. How the archaeal isoleucine tRNA accomplishes this is unknown.