RNA polymerase and the lead ribosome may be physically linked in E. coli by the two-domain NusG protein. This will be demonstrated at the biochemical, structural and genetic levels. We intend to isolate a complex between FL NusG and the 70S ribosome, and to probe its structure using cryo-EM. We will screen a number of mutants for their effects on coupling transcription and translation. Our results indicate that RelA is required for coupling and this will be explored genetically and biochemically. Our studies have implicated tmRNA as the function that uncouples transcription and translation when translation is inhibited. Structural data indicates that tmRNA can displace NusG from the ribosome;we will test this in vitro. NMR and biochemical analyses suggest that NusA can displace NusG from RNAP. This will be tested in a purified transcription system. Chromosome integrity depends on transcription termination. The details of this reaction will be explored. Finally, we find that transcription is linked to repair of a variety of DNA damages. The influence of transcription depends on the type of DNA damage. We will explore the pathways that repair DNA DSBs, DNA adducts, and interstrand crosslinks, and that can remove tightly bound protein from the DNA template.
Transcription and translation are coordinated in all living organisms. This project focuses on how these two synthetic pathways are coupled in E. coli. The project also focuses on how transcription termination in E. coli relates to chromosome integrity and to repair of DNA damage.
|Drogemuller, Johanna; Stegmann, Christian M; Mandal, Angshuman et al. (2013) An autoinhibited state in the structure of Thermotoga maritima NusG. Structure 21:365-75|
|Mooney, Rachel Anne; Schweimer, Kristian; Rosch, Paul et al. (2009) Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J Mol Biol 391:341-58|
|Prasch, Stefan; Jurk, Marcel; Washburn, Robert S et al. (2009) RNA-binding specificity of E. coli NusA. Nucleic Acids Res 37:4736-42|
|Uc-Mass, Augusto; Khodursky, Arkady; Brown, Lewis et al. (2008) Overexpression of phage HK022 Nun protein is toxic for Escherichia coli. J Mol Biol 380:812-9|
|Burmann, Bjorn M; Uc-Mass, Augusto; Schweimer, Kristian et al. (2008) The Y39A mutation of HK022 Nun disrupts a boxB interaction but preserves termination activity. Biochemistry 47:7335-41|
|Cardinale, Christopher J; Washburn, Robert S; Tadigotla, Vasisht R et al. (2008) Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Science 320:935-8|
|Kim, Hyeong C; Washburn, Robert S; Gottesman, Max E (2006) Role of E.coli NusA in phage HK022 Nun-mediated transcription termination. J Mol Biol 359:10-21|
|Washburn, Robert S; Court, Donald L; Gottesman, Max E (2006) Role of an RNase III binding site in transcription termination at lambda nutL by HK022 Nun protein. J Bacteriol 188:6824-31|
|Kim, Hyeong C; Gottesman, Max E (2004) Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues. J Biol Chem 279:13412-7|
|Kim, Hyeong C; Zhou, Jian-guang; Wilson, Helen R et al. (2003) Phage HK022 Nun protein represses translation of phage lambda N (transcription termination/translation repression). Proc Natl Acad Sci U S A 100:5308-12|
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