The long term objective of this proposal is to understand the role played by ribosomal RNA in protein synthesis. The fundamental processes involved in protein synthesis are believed to be the same in all organisms and several studies have indicated direct involvement of highly conserved ribosomal RNA sequences in translation. The goal of this project is to determine the functional significance and structure of a loop in the small subunit ribosomal RNAs of all organisms and located at position 790 in E. coli 16S RNA. A combined genetic, biochemical and structural approach will be employed. In vivo genetics and in vitro biochemistry will be employed to determine function, and NMR will be used to determine the key structural features of the loop. The presence of multiple rRNA genes in most organisms has complicated genetic analysis of ribosome function. To circumvent these problems, a genetic system has been constructed which does not interfere with normal cellular function. In this system, the chloramphenicol acetyltransferase (CAT) reporter message is translated exclusively by plasmid encoded ribosomes which cannot translate normal cellular messages. Consequently, cells containing this construct are chloramphenicol resistant and the level of this resistance is dependent on the amount of functional CAT protein produced by plasmid derived ribosomes. Thus, deleterious rRNA mutations in plasmid encoded ribosomes will inhibit translation of only the CAT message and therefore decrease chloramphenicol resistance without affecting translation of other cellular messages.
Three specific aims are: 1. Identification of key functional elements within the loop. Random mutations will be constructed simultaneously at each of the nine nucleotides in the 790 loop and functional an nonfunctional mutants will be selected. Mutants will be sequenced to identify elements within the loop required for function. Nonfunctional mutants will be analyzed biochemically to determine which aspect of protein synthesis is affected. 2. Identification of sites which functionally interact with the 790 loop. Nonfunctional mutants from 1 will be used to select second-site functional revertants. These will be cloned by complementation and identified. 3. Identification of key structural elements within the 790 loop. Loop structure will be determined using NMR. Key structural elements of the loop will be revealed by structure determination of highly substituted yet functional mutants from 1. Proposed structure/function relationships will be tested by site-directed mutagenesis.
