Research Project
8956485
? DESCRIPTION (provided by applicant): In Escherichia coli a single enzyme is responsible for the synthesis of all transcribed RNA, while multiple auxiliary protein factors associate and functionally interact with the core RNA polymerase complex during different stages of the transcription cycle in order to enable and regulate promoter selection, elongation-related RNA and DNA management, termination of RNA synthesis, and transcript release. A significant body of evidence derived from microbiological and genetic studies indicates that the transition of bacterial cells from active to arrested growth is accompanied by multiple physiological changes, which include promoter-specific changes in the activity of RNA polymerase and alterations in the distribution of transcription complexes, as well as changes in the general architecture of the nucleoid. Despite significant recent advances in methods for protein and small molecule identification/characterization, these growth-specific changes have never been studied through preparative biochemical methods. We propose to carry out a comparative analysis of the proteins and small molecules accessory to transcription complexes obtained from rapidly growing and stationary-phase E. coli cells. In this proposal, we present preliminary data demonstrating the feasibility of such analyses; specifically, we show that there are multiple, distinct growth phase- specific changes in the makeup of proteins accessory to transcription complexes. These growth phase-specific changes likely reflect differences in their macromolecular organization and, possibly, spatial distribution (such as their association with DNA or cellular membranes). We propose (a) to determine the identity of proteins accessory to transcription complexes during rapid and restricted (due to nutrient starvation and/or overcrowding) growth and (b) to carry out a biochemical characterization of the identified proteins and transcription complexes. Specifically, for the key proteins, we propose to carry out LC/ESI-MS-based analyses of small accessory molecules and posttranslational modifications. To demonstrate the feasibility of the latter analyses, we show preliminary data for ESI-MS-based identification of posttranslational modifications in selected RNA polymerase- associated proteins isolated from rapidly growing E. coli cells. E. coli has served as a primary model system for studies of enzymes involved in RNA synthesis; bacterial RNA polymerases and protein factors accessory to the transcription-translation apparatus are the primary targets of numerous drugs and antibiotics. Therefore, advances in understanding the function of the bacterial transcription-translation apparatus are intricately linked to human health. Furthermore, an in-depth characterization of the growth phase-specific changes in the composition of E. coli's transcription complexes would be of fundamental significance for our understanding of bacterial physiology.
