The genes of the adaptive response to DNA methylation damage protect against the lethal and mutagenic consequences of exposure to alkylation damage. The ada gene product acts as a transcriptional activator of this response causing its induction when it becomes methylated as a consequence of its DNA repair activity. The methylated form of Ada (meAda) binds to a region just upstream of the -35 box of the promoters of the adaptive response genes. In the case of the ada and aidB genes, this region is similar to the UP element of the rrnB P1 promoter with respect to its AT richness, its location relative to the core promoter elements at -10 and -35, its ability to be bound by the alpha subunit of RNA polymerase and its apparent enhancer activity. Binding of RNA polymerase to this upstream element can be detected in the presence or absence of meAda. Transcription is activated when meAda binds, triggering RNA polymerase binding to the core promoter elements. I propose to examine this reaction further by addressing the following questions: 1) What is the role of the upstream alpha binding site, comparing its apparent enhancer activity with that of the rrnB P1 promoter s UP element? 2) What interactions between Ada protein and RNA polymerase are required for induction? The aidB gene is an incompletely characterized component of the adaptive response that increases alkylation resistance when it is expressed at high levels. I propose to determine if it is acts by repairing DNA lesions, or if it acts to detoxify specific classes of alkylation agents. The aidB gene is induced not only in response to alkylation damage to DNA, but by several other stresses as well. Induction in response to multiple stresses appears to be a feature common to a number of stress response genes and analysis of the alternative induction pathways will allow the comparison of two induction mechanisms that act at a single promoter. In the case of aidB one of the alternative inducing stresses is exposure to acetate under acidic conditions. This form of induction is dependent upon the rpoS gene product, which encodes the sigma subunit of RNA polymerase, and an uncharacterized positive regulatory element. During the proposed funding period, I propose to characterize this factor further by genetic and biochemical means and to compare its induction mechanism with that of meAda.
