Our long-term goal is to understand the molecular basis of gene expression control. Toward this goal, we have employed the leucine operon, a bacterial genetic system. Our previous studies have demonstrated that the bacterial ilvIH-IeuO-leuABCD: gene cluster is sequentially activated by a novel promoter relay mechanism. As part of the preliminary studies, we have also demonstrated that the leucine operon is transcriptionally silenced by an AT-rich 318 bp upstream element. Final activation of the leucine operon by the promoter relay mechanism involves derepression (reversal of silencing) through binding of a trans-acting factor, LeuO, to the AT-rich element. Toward understanding the underlying mechanism of silencing and its regulation, we have identified a 47 bp AT-rich (85 percent A+T) DNA, AT8, as the critical silencing component within the 318 bp AT-rich upstream element. The mechanism of AT8-mediated gene silencing in bacteria is remarkably similar to that of eukaryotic gene silencing. First, AT8-mediated gene silencing is position specific and gene non-specific. Second, the bacterial histone-like nucleoid structuring protein (H-NS) appears to be required for AT8-mediated gene silencing, which is reminiscent of heterochromatic gene silencing in eukaryotes. Thus, we hypothesize that a local nucleoid structure modification may be responsible for AT8-mediated gene silencing in bacteria. Using the relatively simple (47 bp) AT8 gene silencer as a model system, we propose to identify all trans- and cis-acting players in gene silencing. In addition, we plan to develop an in vitro gene silencing system for more detailed mechanistic studies. We anticipate that the results of our studies in bacteria will shed light on the mechanism of gene silencing in eukaryotes.
