Studies on Transcription Translation coupling and the consequences of its failure in Escherichia coli Modulation of Rho dependent transcription termination by H NS family of proteins

Abstract

The work presented in this thesis was carried out to understand a novel phenomenon newlineof suppression of transcription termination defect associated with rho and nusG mutants by certain mutations in the DNA-binding protein H-NS. H-NS is a highly abundant bacterial protein in E. coli which is the structural constituent of the bacterial nucleoid. It was earlier discovered in this lab that a point mutation in H-NS, E74K suppressed the rho and nusG mutant phenotypes of polarity relief at galEp3, trpE(oc) and also lethality associated with newlineColE1-family plasmids like pACYC184. Another dominant-negative mutant of hns, HNSand#916;64, newlinewhich is a C-terminal truncation of H-NS was also shown to be a strong suppressor newlineof these rho and nusG mutant phenotypes (Harinarayanan and Gowrishankar, 2003). This newlinestudy was undertaken to understand the mechanism of suppression of rho and nusG mutant phenotypes by mutations in hns, particularly H-NSand#916;64. Three different approaches were undertaken to elucidate the mechanism of this suppression namely, (i) obtaining suppressors of H-NSand#916;64 effect in rho and nusG strains by mutagenesis, (ii) testing candidate genes of the H-NS family, that is, hns, stpA, hha and ydgT for suppression of transcription termination defect and (iii) identification of multicopy newlinesuppressors of transcription termination defect in rho and nusG mutants. A total of nine suppressor mutants were chosen to work with and the characterization of eight of these is reported in this study. These are organized as follows.