An organism responds to most environmental insults and developmental cues by altering the transcription of specific genes. Dr. Lis' objective is to achieve a better understanding of the molecular mechanisms by which transcription is modulated in eukaryotes. A unique focal point of this research is the transcriptionally-paused RNA polymerase at the 5' end of the hsp70 gene in uninduced Drosophila cells. Dr. Lis postulates that the progression of polymerase through this pause site is rate-limiting, and therefore the key to regulation. This type of elongational control may be somewhat general, since paused RNA polymerases have been identified at the 5' ends of other Drosophila and human genes. The first half of this proposal focuses on the heat shock genes of Drosophila and two distinct processes in gene activation: potentiation and activation. Potentiation is characterized by the disruption of chromatin structure at the promoter and the generation of a promoter-paused RNA polymerase. Dr. Lis proposes to investigate the interplay of promoter elements that are responsible for potentiation by mutagenesis coupled with assays of protein-DNA interactions in vivo. In particular, the interactions between GAGA factor, TFIID, paused polymerase and their respective sequence elements will be studied. Activation occurs subsequent to potentiation and is mediated by heat shock factor (HSF). This activation stimulates resumption of elongation by the paused polymerase. Alternative models for the regulation of this rate-limiting step will be tested. Polymerase pausing and the elongational """"""""escape"""""""" from this pause during heat shock will be examined on an hsp70 promoter where HSF interaction with its sequence elements (HSEs) is disrupted either by mutations in HSEs or by expressing a dominant negative mutant of HSF. Dr. Lis plans to test the ability of purified HSF and other activators to stimulate elongation from the pause, assayed in intact nuclei or nuclear extracts. He will attempt to disrupt the proposed tether that retains the paused polymerase on the promoter by protease cleavage at engineered sites and by altering the rotational phasing of interacting partners. The second half of this proposal explores the use of yeast to evaluate models derived from the analysis of Drosophila transcriptional control. Dr. Lis proposes to characterize polymerase-promoter interactions in yeast using both in vivo footprinting and protein-DNA crosslinking. Possible interactions between factors at the promoter will be examined in vitro using affinity chromatographic methods. Mechanistic models for transcriptional regulation derived from these in vitro experiments will then be tested by mutagenizing proteins found at the promoter and determining the effects on the promoter's protein-DNA architecture.
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