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 processing of intron-containing pre-tRNAs in Haloferax volcanii, a halophilic archaeon.
The specific aims of this proposal are: Characterization of in vitro sRNP system for site-specific modifications in H. volcanii pre-tRNATrp; Characterization, in vivo, of the production of 2'-O-methylcytidine (Cm) and 2'-O-methyluridine (Um) at positions 34 and 39, respectively, of the tRNATrp in H. volcanii and study, in vivo, the relationship between these modification reactions and pre-tRNA splicing; and Determination of the effect of Cm modification at the wobble position of tRNA on the accuracy of translation in H. volcanii. Mechanism of 2'-O-methylation of H. volcanii pre-tRNATrp nucleotides, in vitro, will be studied by using recombinant box C/D RNP core proteins and H. volcanii cell extracts along with 32P-labeled T7 RNA polymerase generated pre-tRNAs and their introns. Binding of core proteins to normal and variously modified substrates will be determined by gel-shift assays. Production of the 2'-O-methylated nucleotides will be determined by thin layer chromatography (TLC) of the RNase T2 or nuclease P1 digests of the RNAs. A modified version of tRNATrp gene will be used to study, in vivo, box C/D guided modifications reactions and their relationship to splicing. The tRNA product of this modified gene and normal genomic gene can be distinguished. Products of variously mutated versions of this modified gene will be characterized in in vivo studies. Extracts of the cells containing mutated forms of this modified gene will also be used for the in vitro methylation studies. Role of the intron in wobble base modification of tRNA and its effect on the accuracy of translation will be tested by using specifically modified reporter gene and an intron-deleted tRNA gene. The tRNA products of this intronless gene will be characterized for the presence or absence of specific Cm and Um modifications by separating their RNase T1 digests by denaturing gels or fingerprinting, and determining the composition of the appropriate oligonucleotides by TLC.
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