Mobile genetic elements have been found in a wide variety of procaryotic and eucaryotic organisms. They are widely thought to represent one of Nature's principal tools for the rearrangement of genetic material. Our long term goal is to understand in detail how the transposition of mobile genetic elements is controlled. As a step toward this goal, we will study the details of a novel regulatory mechanism in the bacterial insertion sequence IS10: the post-transcriptional control of IS10 transposase gene expression by a small IS10-specified anti-sense RNA. We term this control """"""""multicopy inhibition"""""""", and our working model is one where the anti-sense RNA binds to and inhibits translation of the transposase mRNA. This problem will be studied both genetically and biochemically. Using genetic means, we will introduce point mutations into IS10, and screen for mutants that are altered in multicopy inhibition. We expect to find two types: those that alter the quality and/or quantity of the anti-sense RNA, and those that alter the site, in the transposase mRNA, at which the anti-sense RNA acts. Mutants will be subjected to a battery of sophisticated genetic tests to determine how the mutations affect various aspects of IS10 behavior, including transposition, transposase function production, complementability, and multicopy inhibition. The transcriptional activity of these mutants, and the character of the transcripts they produce, will be studied in vivo, with particular attention given to the fate of these transcripts during multicopy inhibition. We will attempt to express and isolate large amounts of wild-type and mutant IS10 transcripts, to be used for in vitro studies on transcript binding. We will examine the hypothesis that multicopy inhibition is exerted at the level of translation of the IS10 transposase by studying translation in vitro, initially with the Weissbach di-peptide synthesis assay. By isolating and characterizing mutations in the E. coli host, we will attempt to determine which cellular processes participate in multicopy inhibition, and at what level. Particular emphasis will be placed on possible host functions that promote or retard pairing between complementary RNAs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035322-03
Application #
3287862
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-07-01
Project End
1988-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Sussman, J K; Simons, E L; Simons, R W (1996) Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo. Mol Microbiol 21:347-60
Ma, C K; Kolesnikow, T; Rayner, J C et al. (1994) Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation. Mol Microbiol 14:1033-47
Pepe, C M; Maslesa-Galic, S; Simons, R W (1994) Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack. Mol Microbiol 13:1133-42
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Ma, C; Simons, R W (1990) The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO J 9:1267-74
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