The archaebacteria are distinct from the eubacteria and the eukaryotes. Though the basic life processes are similar in the three kingdoms, in details of molecular features and various functional mechanisms, the archaebacteria are either unique or resemble one or the other of the remaining two kingdoms. There are two main divisions of archaebacteria: (i) methanogen-extreme halophiles, and (ii) sulfur-utilizing thermophiles. The latter division more closely resembles eukaryotes than does the former. Therefore, the study of archaebacterial systems, besides having a comparative phylogenetic importance, could also be considered as a study of a eukaryote-like system at a simpler (prokaryotic) level, which might have evolved to a more complex (eukaryotic) level. The long term goals of this study are to understand the structure and function of transfer RNAs and the organization and expression of tRNA genes in archaebacteria.
The specific aims of this proposal are the isolation and sequencing of Sulfolobus sp. tRNAs, characterization of modified nucleosides in archaebacterial tRNAs, determination of the sequences of Halobacterium volcanii tRNA genes and their flanking regions, determination of RNA polymerase binding sites in archaebacterial genes to identify promotor regions, and development of an in vitro RNA processing system for archaebacterial gene transcripts. The tRNAs will be sequenced by a combination of two-dimensional paper electrophoresis of RNAse digests of uniformly 32P-labeled tRNAs and gel sequencing methods using end-labeled tRNAs. Modified nucleosides will be identified by various electrophoretic, chromatographic, mass spectrometric and NMR techniques. The tRNA genes will be cloned in plasmid vectors and sequenced by dideoxy termination and chemical methods. RNA polymerase binding sites in promotor-containing DNA will be determined by the DNAse I """"""""foot printing"""""""" technique. Pilot studies will be done to identify an RNA processing activity in crude cell extracts using either natural precursors or in vitro SP6 or T7 polymerase-transcribed RNAs from archaebacterial tRNA genes. Then attempts will be made to define and characterize the components involved in this processing system.
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