UC Riverside

The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed, it is critical to the control of gene expression in bacteria. Despite the recognition of its importance to the cell’s physiology and metabolism, a detailed and comprehensive understanding of its mechanistic underpinning has remained elusive. In my thesis, I aimed to uncover potential routes of crosstalk between RNA polymerase and ribosome, the key players of transcription and translation, respectively. In my in vitro work, I identified direct interactions between both of these nanomachines as potential element for the regulation of the coupling of both processes. Using several methods I was able demonstrate the existence and specificity of this interaction and in collaboration with my colleagues from the Blaha and Wang laboratories, I was able to narrow the RNA polymerase binding interface on the ribosome to the head region of the small ribosomal subunit. All this progress in our understanding of transcription-translation coupling depended on a reliable method of separating free from ribosome-bound RNA polymerase. To this end I have established a reliable analytical procedure using sucrose gradient centrifugation followed by SDS polyacrylamide gel electrophoresis. Recently, I improved this procedure by replacing the sucrose gradient centrifugation with native gel electrophoresis, thus reducing the time and the amount of material required for the analysis of the interactions between RNA polymerase and ribosomes. To place my finding of the direct interaction between RNA polymerase and ribosomes in context of the resurgence of interest in the mechanism of coupling, I have included in this thesis the review I co-authored that summarizes the recent literature of the field. In this review, we integrate the presented data of the literature and of my work into a dynamic model of transcription-translation coupling in which the interactions between RNA polymerase and ribosomes are repeatedly formed and broken. The careful comparison of the effects of the direct interactions between RNA polymerase and ribosomes with those mediated by transcription factors NusG and RfaH leads us to propose two distinct modes of coupling: a factor-free and a factor-mediated coupling.