The proposed research program will be directed toward the establishment of a method for the genetic analysis of eukaryotic DNA/protein interactions in E. coli. Experiments will make use of the interaction of the internal control region (ICR) of 5S ribosomal RNA genes with the 5S-specific positive transcription factor IIIA (TFIIIA) as a model case. The approach involves the construction of bacterial genes that are placed under negative control of TFIIIA in experiments in which TFIIIA functions as a repressor. Vectors are described for the production of TFIIIA in E. coli and for the use of the 5S ICR as an """"""""operator"""""""" sequence to control a selectable bacterial gene. Point mutants in the 5S ICR that have a lower affinity for TFIIIA will be selected on the basis of their oc phenotype. Others will be prepared by site-directed mutagenesis. Mutants in TFIIIA that have lost sequence-binding specificity or that have gained altered specificity and bind to the mutant ICRs will be selected, based on the TFIIIA """"""""repressor"""""""" function. A modification of the approach will be used to select for colnes that encode TFIIIA from cDNA libraries and also for clones encoding other sequence-specific DNA binding proteins in the absence of information concerning protein structure or identity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034818-01
Application #
3286449
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106