The proper expression of a gene is often dependent on the chromosomal position of the gene. This position effect is poorly understood at the molecular level. We have demonstrated that plasmid-borne leu-500 minimal promoter cannot be activated in topoisomerase I (topA) mutants unless adjacent transcription (located within 250 bp) occurs in a direction away from the leu-500 promoter (short range promoter interaction). To search for a potential upstream promoter responsible for activation of leu-500 in the chromosomal context, we have identified the ilvIH promoter, located 1.9 kb upstream of leu-500 promoter (long range promoter interaction). In contrast to the short range promoter interaction, which is abolished by DNA sequence insertions, the long range promoter interaction is in fact mediated by the intervening sequence. The intervening sequence appears to play a crucial role(s) in regulating the expression of the leucine operon. A part of this intervening sequence itself in the absence of the ilvIH promoter seems to repress the wild- type leucine promoter. The repressed leucine promoter is fully depressed upon inclusion and activation of the ilvIH promoter located 1.9 kb upstream. Apparently, the proper expression of the bacterial leucine operon is dependent on transcriptional activity of the ilvIH operon and the 1.9 kb intervening sequence between the two operons. Since transcription is one of the most important fundamental biological processes, its impact on DNA structure and function deserves careful investigation. Toward this end, we propose to study the mechanism of long range interaction between two promoters using the bacterial leuABCD and ilvIH operons as a model system. We hypothesize that the long range interaction between a pair of divergently arrayed promoters is mediated by a complex positively and negatively acting regulatory processes involving a relay of DNA supercoiling by the DNA sequence located between the two promoters. To dissect the complex communication between the two promoters, we plan (A) to determine the effect of transcription-mediated DNA supercoiling in modulating the long range promoter interaction, (B) to identify cis- and trans-acting genetic element(s) responsible for the long range interaction between ilvIH and leuABCD promoters, and (C) to test whether the long range promoter interaction is mediated by a depression mechanism. This research is likely to impact on various aspects of medical genetic research as the proposed studies will reveal a novel gene expression regulatory mechanism involving transcription- mediated DNA supercoiling.
