The objectives of this proposal are to understand: (1) the relationship between the structure and the many distinctive properties of E. coli initiator tRNA and (2) the molecular mechanisms underlying its specific interactions with components of the translational machinery. An important question is the molecular basis of the highly specific recognition of the tRNA by Met-tRNA transformylase. A combination of structural, biochemical, and genetic approaches will be used to study this. These include (I) NMR spectroscopy to analyze the structure of the tRNA substrate, (ii) crystallography to determine the structure of the protein and the tRNA-protein complex, (iii) investigation of the topology of interaction of the protein with the tRNA using crosslinking experiments, protection experiments and by examining suppressor mutations in the protein that compensate for defects in formylation of mutant tRNAs, and (iv) site-specific mutagenesis to identify amino acids important in the protein for tRNA selection and function. Similar approaches will be used to study interactions of tRNA with other proteins, in particular, in vivo selection and analysis of suppressor mutations in IF2, IF3, or other chromosomal genes. Such work could provide information on interactions between the initiation factors and the initiator tRNA and could lead to identification of new genes involved in translational initiation. Work on identification of intermediates in translation initiation in vivo will continue. Questions of specific interest are: (I) is IF2 a carrier of fMet-tRNA to the ribosome? (ii) Does the 30S ribosome bind first to the initiator tRNA and then to the mRNA or vice versa? (iii) Are the requirements in an initiator tRNA for translational reinitiation the same as for de novo initiation? Finally, the role of the All:U24 base pair unique to eubacterial initiator tRNAs in initiation will be studied along with questions of why introduction of the base pair to an E. coli elongator methionine tRNA prevents accumulation of the tRNA in vivo.
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