Studies of gene regulation help us in understanding cellular metabolism, growth, and differentiation. Post-transcriptional RNA processing is one of the several ways by which gene expression is regulated. The long-term objective of this AREA application is to characterize RNA processing in archaea, especially in the halophilic and then thermophilic archaea. Archaea are prokaryotes like bacteria; yet in several of their molecular features, they resemble eukaryotes more than the bacteria. Therefore, these studies besides providing information about the archaeal systems will also help in understanding the corresponding eukaryotic systems.
The specific aims of this proposal are: Determination of the source of splice junction phosphate in Haloferax volcanii spliced tRNAs; Purification of H. volcanii tRNA splicing ligase; Determination of the mechanisms of ligation and ligase binding in tRNA splicing in H. volcanii; Determination of the presence or absence of 2'-O-r;ethylcytidine (Cm) modification at the wobble position of in vivo generated products of recombinant intron-deleted tRNA genes in H. volcanii; and Determination of the relationship among tRNA intron splicing, modifications at the wobble position of corresponding tRNAs, and accuracy of translation in H. volcanii.
These aims are planned to determine the mechanism of pre-tRNA splicing and the reasons for the existence of tRNA introns in halophilic archaea. H. volcanii cell extracts containing splicing ligase activity will be used with unlabeled T7 RNA polymerase produced transcripts and [gamma 32P]GTP in one set of experiments and labeled transcripts (using [alpha -32P]GTP) and unlabeled ATP in another set. The products of the reaction will be analyzed by specific combinations of RNase Tl, T2, nuclease P1 and venom phosphodiesterase digestions to determine the source of splice junction phosphate. Ligase will be purified by the methods in which high concentrations of salts can be maintained continuously. Partially purified ligase will be used to ligate modified in vitro produced exons, in the studies determining mechanism of ligation. A potential role for the intron in modification at the wobble position of tRNA, and the effect of the modification on the accuracy of translation will be tested by genetic methods, using specifically modified reporter protein systems and intron-deleted tRNA genes. The tRNA products of these intronless genes will be characterized for the wobble modification by a specific combination of RNase T1 and T2 digestions.
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