RNA polymerase and the ribosome are two of the most well-studied macromolecules in all of molecular biology. Most research done on RNA polymerase and the ribosome has been done in isolation from each other. However, the cellular processes the RNA polymerase and ribosome carry out, transcription and translation, respectively, occur in the same cellular compartment in bacteria. In fact, the process of transcription and translation are coupled. Despite a detailed understanding of various aspects of transcription and translation, the coupling of both processes has remained enigmatic. This includes the interaction interface between the RNA polymerase and the ribosome, and between the RNA polymerase and the ribosomal subunits. Furthermore, we know how only a few ribosomal proteins influence transcription, a need for additional detailed studies are required to elucidate their potential effects on transcription during coupling. In my thesis, I first aimed to determine the interaction interface between RNA polymerase and the ribosome and between the RNA polymerase and the small and large ribosomal subunit. To do this, RNA polymerase was cross-linked with either the ribosome or one of the ribosomal subunits. The mixture was run on an SDS polyacrylamide gel, the crosslinked protein bands were excised from the gel, and the crosslinked proteins were identified in collaboration with Dr. Wang groups in the Chemistry department at UC Riverside. Based on this analysis we could identify several proteins on the ribosome that may participated in different interactions between the RNA polymerase and ribosome during transcription-translation coupling. For three of these ribosomal proteins, I determined their effects on transcription. Each ribosomal protein exerted a unique and unexpected effect on transcription. They all slowed the RNA polymerase at the his pause sequence. Two of them even hampered factor-mediated transcription termination, while one bound directly to core RNA polymerase. To enable a smooth follow-up of my initial studies of the ribosomal proteins' effect on transcription, I have included an extended appendix that details all aspects critical to performing these transcription assays successfully.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsDABG is a soluble NAD-dependent formate dehydrogenase and a member of the DMSO reductase family of molybdoenzymes and NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex from the cytosolic FdsDABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 – both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 cluster B6 in FdsB and Fe2S2 cluster G7 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, not previously observed to participate in NADH-mediated reduction of the FdsDABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the G7 Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path electron transferring chain of iron-sulfur clusters connecting the FMN in the FdsB subunit with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN; the structure of FdsDABG in Rhodobacter capsulatus shows a much further binding of the nicotinamide ring relative to the FMN. Regardless, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

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.