Structure, Function, and Genetics of Ribosomal Components

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This project will focus upon the structure, function and genetics of protein and RNA components of the prokaryotic ribosome. (1) The structural features within E. coli 16S and 23S rRNAs that account for their specific interaction with r- proteins S8 and L1 during ribosome assembly will be probed by site-directed mutagenesis and protein-rRNA cross-linking. In addition, the minimum rRNA segment required for protein binding will be determined to facilitate their detailed physical characterization in the future. (2) Over 40 mutants of E. coli r-protein S8 have been isolated on the basis of their inability to translationally regulate spc operon expression. The metabolic and physically stability of these proteins will be examined, and their RNA-binding properties evaluated. 2D NMR analysis of wild-type and mutants S8 will be used to define the structure of S8, and its RNA-binding domain, at high resolution. (3) Operons encoding components of the translation and transcription apparatus in several thermoacidophilic archaebacteria will be isolated and sequenced. Various approaches will be employed to gain insights into how the expression of these transcription units is controlled. The results will provide a better understanding of the evolution of gene regulation, as well as of gene organization, in this unusual group of organisms.