9600955 Arndt Regulated transcription initiation by RNA polymerase II requires the involvement of several classes of transcription factors. As the initial event in preinitiation complex assembly, binding of TBP to the promoter is a pivotal step in transcription. Recent studies suggest that the general transcription factor TFIIA directly and indirectly affects the formation and stability of the TBP-TATA complex. To dissect the functions of TFIIA in vivo, an extensive genetic analysis of the genes that encode the two subunits of yeast TFIIA, TOA1 and TOA2, will be performed. To date, few mutations in these genes have been identified. Mutations that cause informative mutant phenotypes, espe2'd Letter SM to MC.cially those that might indicate specific transcriptional defects will be obtained by introducing random mutations into TOA1 and TOA2 by PCR followed by genetic screening. The effects of these mutations on TFIIA function will be determined by various biochemical methods. These experiments will provide a detailed structure-function picture of TFIIA and will identify valuable mutants to be used in subsequent genetic and biochemical studies. Studies on the properties and TFIIA suppression of TBP activation-defective mutants have led to the suggestion that promoters may differ in the degree to which they require TFIIA for normal regulation. This idea will be tested by depletion of TFIIA levels in vivo by conditionally expressing TOA1 and TOA2. The transcriptional effects of TFIIA depletion on different classes of activated genes and on constitutive genes will be measured and compared. In highly purified in vitro systems, TFIIA can stabilize the binding of TBP to the TATA box. Genetic studies suggest that TFIIA may perform a similar function in vivo. Testing this hypotheses directly will be done by using genomic footprinting methods to monitor TATA box occupancy in TBP mutant strains that express high levels of wild-type TFIIA or mutant forms of TFIIA TATA box occupancy at several inducible promoters will be examined. The teaching goals are to provide students with a solid foundation in the concepts and practical applications of genetic analysis. Two new undergraduate-level Microbial Genetics courses, a lecture and a laboratory, have been recently developed. These courses will be improved by adding problem sets, instituting problem-solving workshops, and integrating current research topics and approaches into lectures and laboratory exercises. The goal is to provide beginning and advanced graduate students with a current understanding of several fields in biology, including transcriptional regulation and yeast genetics. Recent research discoveries will be introduced into lectures and discussions; scientists from outside the University will meet with students for discussions as well. ***
