The long term objective of this proposal is to examine in detail a postulated biochemical pathway including the T4 induced enzymes anticodon nuclease, polynucleotide kinase and RNA ligase. These enzymes are functionally related, presumably operating in host tRNA cleavage and reprocessing. These assumptions rest on (i) the specific manifestation of anticodon nuclease in E. coli CTr5x, a host restrictive to kinase and ligase mutants, (ii) accumulation of tRNA fragments generated by the anticodon nuclease in the mutant infections, (iii) lack of anticodon nuclease in pseudorevertants of kinase and ligase mutants able to grow on the restrictive host and (iv) cotransduction into a permissive host of the abilities to restrict kinase and ligase mutants as well as to express tRNA species vulnerable to the anticodon nuclease. The specific goals are: (i) isolation and characterization of the anticodon nuclease, (ii) cloning of the restrictive host gene and its structural and functional analysis; (iii) detection in vivo of the reprocessing substrate, intermediates and product, and (iv) reconstitution studies with purified enzymes and substrates. The anticodon nuclease will be isolated using the specific cleavage of E. coli CTr5x tRNAs as an assay and mutants lacking the anticodon nuclease for complementation. Cloning the T4 RNA ligase suppressor gene which specifies the anticodon nuclease will be attempted to generate an anticodon nuclease overproducer. The restrictive host gene will be cloned into a plasmid vector using the vulnerable tRNA fragments as hybridization probes and conferral of restriction towards kinase and ligase mutants as a selectable marker. Purifying the anticodon nuclease and isolation of the vulnerable tRNA genes will facilitate in vivo and in vitro analyses of the reprocessing pathway. These studies may define physiological roles for T4 anticodon nuclease, polynucleotide kinase and RNA ligase and reveal a novel translational control mechanism through reprocessing of mature tRNA.
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