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 (encoding chloramphenicol acetyltransferase) in Gram-positive bacteria. Induction of one gene, cat-86, has been shown to be due to activation of mRNA translation. This appears to result from the ability of Cm to stall ribosomes translating a cat-86 regulatory leader peptide; a stalled ribosome is thought to disrupt a downstream 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 anticipated that these studies will clarify and extend our understanding of the mechanism of action of a ribosomally targeted antibiotic. It is also expected that these studies will show the breath 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 spatial relationship between the stall site and the regulated RNA secondary structure will be identified. Uncommon examples of apparently Cm-insensitive translation exit in bacteria. Do these result from the fortuitous absence of a Cm stall site? Certain species of the soil bacterium genus Bacillus are naturally resistant to the antibiotic chloramphenicol by virtue of their ability to synthesize an enzyme which inactivates the drug. The synthesis of the inactivating enzyme is specifically inducible by the drug, i.e., the bacteria make the enzyme only when they encounter the drug. Dr. Lovett's earlier studies have shown that a drug-induced change in the behavior of ribosomes, the organelles which synthesize proteins, is a critical part of the mechanism of the induction of the inactivating enzyme. This is a very unusual mechanism for the regulation of gene expression, and Dr. Lovett proposes in this study to work out many of the still obscure details of this mechanism. Apart from insight into a novel regulatory mechanism, this work should also yield new insight into how the function of ribosomes is controlled.
