The primary goal of this proposal is to elucidate the mechanism of action of a model antiterminator, the N protein encoded by temperate phage lambdal. N is a small, modular protein that interacts with a specific genetic signal (called nut) on the phage genome and a number of host proteins (called Nus factors) to modify host RNA polymerase to a termination-resistant mode. The current working model proposes that an arginine motif present in N binds the boxB RNA hairpin encoded by nut, bringing N in close contact with the target polymerase. N then captures polymerase through mRNA looping and collaboration of an adapter host factor (NusA), and leads RNA polymerase through downstream terminators by being an operon-specific subunit. Additional host factors interact with conserved RNA signals and N to facilitate both the assembly as well as persistent antitermination. The proposed experiments will combine genetic, biochemical and biophysical approaches to accomplish several major objectives. The first goal is to carry out several lines of mechanistic studies to rigorously test various predictions of the model, further dissecting the role of the core components in termination-suppression and elucidating the underlying molecular mechanisms. The second goal is to isolate the missing host factor(s) and their genes by independent genetic and biochemical approaches and elucidate their function in assembly of the N-antitermination complex, persistent antitermination, and in switching phage development from lysogenic to the lytic mode. The third goal is to study N-boxB interaction with molecular genetic and biophysical approaches to decipher the chemical principles that govern RNA-protein interaction. The fourth goal, a new direction of the project, is to identify cellular genes that are activated by N-like antitermination mechanism, and isolate potential cellular homologues of N. These studies should provide fundamental information on the structure, function and regulation of the prokaryotic transcription apparatus, should illuminate the function of RNA signals in transcription control, and should reveal aspects of the molecular basis of sequence-specific recognition of RNA signals by regulatory proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Connecticut
Schools of Dentistry
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Das, Asis; Garcia Mena, Jaime; Jana, Nandan et al. (2003) Genetic and biochemical strategies to elucidate the architecture and targets of a processive transcription antiterminator from bacteriophage lambda. Methods Enzymol 371:438-59
Kulish, D; Lee, J; Lomakin, I et al. (2000) The functional role of basic patch, a structural element of Escherichia coli transcript cleavage factors GreA and GreB. J Biol Chem 275:12789-98
Toulme, F; Mosrin-Huaman, C; Sparkowski, J et al. (2000) GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming. EMBO J 19:6853-9
Garcia-Mena, J; Das, A; Sanchez-Trujillo, A et al. (1999) A novel mutation in the KH domain of polynucleotide phosphorylase affects autoregulation and mRNA decay in Escherichia coli. Mol Microbiol 33:235-48
Rees, W A; Weitzel, S E; Das, A et al. (1997) Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda. J Mol Biol 273:797-813
Van Gilst, M R; Rees, W A; Das, A et al. (1997) Complexes of N antitermination protein of phage lambda with specific and nonspecific RNA target sites on the nascent transcript. Biochemistry 36:1514-24
Das, A; Pal, M; Mena, J G et al. (1996) Components of multiprotein-RNA complex that controls transcription elongation in Escherichia coli phage lambda. Methods Enzymol 274:374-402
Liu, K; Zhang, Y; Severinov, K et al. (1996) Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA. EMBO J 15:150-61
Rees, W A; Weitzel, S E; Yager, T D et al. (1996) Bacteriophage lambda N protein alone can induce transcription antitermination in vitro. Proc Natl Acad Sci U S A 93:342-6
Das, A; Barik, S; Ghosh, B et al. (1996) Immunoprinting: a technique used to study dynamic protein-nucleic acid interactions within transcription elongation complex. Methods Enzymol 274:363-74

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