The primary objective of this investigation is to determine how various protein factors act to regulate gene expression in Escherichia coli and bacteriophage-infected E. coli at the level of RNA transcription. The major emphasis will be on the structure, function and mechanism of action of the rho transcription termination protein, on how rho action is modulated by NusA protein and on sequences required for the actions of rho and NusA in termination.
The specific aims are: to identify sequences and specific nucleotides required for termination of transcription at the tR1 site of bacteriophage lambda and for lambda gene N antitermination function; to analyze the interactions between rho and NusA proteins with cro mRNA by binding and ATPase activation measurements; to identify rho utilization (rut) sites for the intragenic lacZ terminator (tlacZ1) and for lambda tL1; to analyze the distribution of intragenic transcription termination sites in E. coli genes and to demonstrate that these terminators have a physiological function; and to localize functional domains for interactions with ATP, RNA, NusA and other rho subunits along the sequence of rho protein. The majority of the analyses will be made using in vitro assays with highly purified components. Site-directed mutagenesis techniques will be employed to create mutations in gene sequences that are likely to govern the action of rho, NusA and the N-antitermination mechanism. DNA sequencing will be used to identify mutational alterations. Transcription of specially constructed DNA templates with T7 RNA polymerase will be used to prepared RNA for interaction studies. Photochemical, chemical and enzymatic methods will be used to define sites of contact between protein in RNA. Gel electrophoresis techniques will be used for assays for termination function in vitro and for determining sites of contact between protein and RNA. Fusion of DNA segments to galK gene expression plasmids will be made to assay for in vivo function of terminators. The insights gained from understanding the fundamental mechanisms of gene expression will help in understanding the etiology of diseases, such as cancers and viral infections, that result from alterations of normal gene expression.
