Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Broad, long-term objectives of this AREA application are to characterize various RNA processing events in archaea. Both archaea and bacteria are prokaryotes; yet archaea exhibit several molecular features resembling eukaryotes. This proposal specifically deals with pre- tRNA splicing in Haloferax volcanii, a halophilic archaeon. A long-term goal is to also clone and overexpress the gene for H. volcanii splicing ligase.
The specific aims of this proposal are: Purification of H. volcanii tRNA splicing ligase; Determination of the structural requirements for the substrates of H. volcanii splicing ligase; Characterization of H. volcanii splicing ligase reaction; Determination of the presence or absence of 2'- O-methylcytidine (Cm) modification at the wobble position of the tRNA produced in vivo in H. volcanii by an intronless tRNA gene; and, Determination of the effect of Cm modification at the wobble position of tRNA, on the accuracy of translation in H. volcanii. H. volcanii splicing ligase is inactivated under low salt concentrations containing solutions. Therefore, the ligase will be purified by the methods where high concentrations of sodium/potassium salts are maintained continuously. Certain procedures where ammonium sulfate or organic solutes replace these salts, will be used for the purification. Several modified in vitro produced and commercially available substrates will be tested in ligation reactions for determining the structural requirements for the substrates of ligase. Ligase-substrate binding will be characterized by gel-shift assays using some of these substrates. Various different substances and modified substrates will be examined for their effect on the ligase reaction. Role of the intron in modification at the wobble position of tRNA and its effect on the accuracy of translation will be tested by genetic methods, using a specifically modified reporter protein system and an intron-deleted tRNA gene. The tRNA product of this intronless gene will be characterized for the presence or absence of Cm modification at the wobble position of the tRNA, by RNase T1 fingerprinting.
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