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 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 benzoylphenylalanine 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, additional experiments are necessary and are proposed in here. 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. The question of whether the specificity of ochre suppressor tRNAs is determined by the nature of the modified base in the anticodon and/or the ribosome will also be addressed. A further goal is to generate archaeal strains carrying suppressor tRNA genes in the hope of facilitating archaeal and archaeviral genetics. (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 the focus of studies proposed in here.
Protein synthesis, which involves the translation of genetic information present in the form of nucleotide sequences in a messenger RNA into amino acid sequences of a protein, is one of the important steps in gene expression. We are interested in the role that a class of molecules called initiator tRNAs play in this process. Because many of the antibiotics, which we currently use, target the protein synthesis machinery, an understanding of how the molecules involved in this process perform their function could lead to the development of new antibiotics.
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