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.
| Campbell Jr, F E; Setzer, D R (1991) Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase. Mol Cell Biol 11:3978-86 |
