Chloramphenicol (Cm) inhibits bacterial growth by blocking peptide elongation during translation on 70S ribosomes. Cm also acts as an inducer of the expression of cat genes (specify chloramphenicol acetyltransferase) in Gram-positive bacteria. Induction of one gene, cat-86, has been shown to e due to activation of mRNA translation. This results from the ability of Cm to stall ribosomes translating a cat-86 RNA stem-loop that sequesters the cat-86 ribosome binding site. The long term goal of this proposal is to explain the mechanism which allows Cm to activate cat-86, and other inducible cat genes. It is also expected that these studies will show the breadth of the principles of the attenuation regulatory model in controlling gene expression. By use of site directed mutagenesis and recombinant DNA technology it is now possible to determine whether the proposed Cm stall site is an mRNA or protein sequence, and the sequence of the site. The special relationship between the stall site and the regulated RNA secondary structure will be identified. Uncommon examples of apparently Cm- insensitive translation exist in bacteria. Do these result from the fortuitous absence of a Cm stall site? Erythromycin probably stalls ribosomes at a site different from Cm. It has been possible to convert cat-86 to an erythromycin inducible gene, and this should provide a novel approach to identify the presumptive erythromycin stall site. cat-86 mRNA is cleaved at two locations by an apparent endonuclease in B. subtilis. We believe we have identified one of the sites susceptible to cleavage. The sequence this site is similar to exon-intron junctions in eukaryotic mRNA. Experiments to characterize this endonuclease activity are proposed. An inducible cat gene from a gram-negative and an inducible streptomycin- resistance gene from a gram-positive will be characterized with respect to the induction mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042925-05
Application #
2181738
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1989-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1996-06-30
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21250