Base modification in tRNA represents the find tuning of a crucial process in cellular metabolism - the faithful translation of messenger RNA into protein. The modification enzymes are unique in that their substrates are highly structured tRNA molecules; thus, they provide an opportunity to study protein-RNA interactions in the context of mechanistic enzymology. The long range goal of this work is to understand the enzymatic mechanisms of thiol modification at two specific sites in E. coli tRNA, uridine 8 (U8) and adenosine 37 (A37), that are implicated in regulatory events in bacterial metabolism. In addition, these enzymes will be used to explore RNA-protein interactions in an effort to understand the basis for their observed specificity at the nucleotide level.
The specific aims of the proposed work are as follows: (1) Enzyme assays will be developed to allow the purification of the modification enzymes. The enzymes will be purified to homogeneity, using affinity chromatography of immobilized RNA fragments derived from substrate specificity studies. The structural genes will be cloned from E. coli using sequence information from the purified proteins. (2) the mechanism will be investigated by which sulfur is transferred to the tRNA bases, in each case from cysteine via different cofactors and presumably different mechanisms. (3) The role of iron dependence in the thiolation of A37 will be explored through the development of specific inhibitors of this step and observation of the metabolic effects of such inhibition as compared to previously described genetic mutants. (4) The tRNA substrate specificity of the enzymes will be elucidated using in vitro selection techniques that explore the primary, secondary and tertiary structure determinants for activity as a substrate. The thiol modifications will be used as chemical tags to retrieve active molecules from mutagenized libraries of E. coli tRNAPhe. Specific relevance to medical issues lies in the fact that the A37 modification is thought to act as a sensor in bacteria for conditions of low iron availability. Such conditions arise in host (e.g., mammalian) fluids inhabited by enteric pathogenic bacteria. In addition, the knowledge gained from this work is directly relevant to the current study of molecular recognition events between RNA and proteins, such as retroviral assembly and RNA processing and transport, that are involved in many disease related processes.
