Robert G. West - US grants

We seek to determine the molecular mechanisms involved in positive and negative control of expression of eukaryotic genes transcribed by RNA polymerase II. We exploit the genetic attributes of GAL (galactose metabolism) gene expression in the yeast Saccharomyces cerevisiae to achieve our goal. We are studying three distinct molecular mechanisms that govern the expression of the adjacent and divergently transcribed GAL1 and GAL10 structural genes. These mechanisms include: repression, antirepression and positive control. Together they comprise a genetic switch that moderates the abundance of the GAL1 and GAL10 gene products over a range of four orders of magnitude. Our first objective is to determine the molecular basis of repression of GAL1 and GAL10 caused by sequences located in the upstream activation site (UASG) region. The function of repression is to reduce the basal level of transcription of these genes by a factor of 1000 fold. We have determined that this particular form of negative control is independent of GAL80 gene product, a previously characterized negative regulator of GAL gene expression, and GAL4 gene product, a positive regulator of the GAL structural genes. Our present efforts are directed at characterizing the putative repressor and its binding sites in UASG, and determining the molecular mechanism of repression. Secondly, our data show that carboxy-terminal truncations of GAL4 product, retaining the DNA-binding domain but lacking the positive control function, can overcome repression. This may suggest that GAL4 product normally competes with a repressor molecule to bind a UASG. We wish to determine the biochemical mechanism that favors activation over repression. Thirdly, we have constructed specific mutations of the GAL4 gene to localize domains rsponsible for its positive control function. Our ultimate goals are to procure missense mutations in such a domain(s) and to obtain trans-acting suppressors of those mutations. By characterizing the latter we may be able to identify the property which causes GAL4 product to induce transcription of the GAL structural genes. Combined, the above projects will reveal several key mechanisms of negative and positive transcriptional control, and how the two may be coordinated to regulate different levels of expression of eukaryotic genes.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of availability data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.