In this competing continuing research grant application we propose to extend our ongoing studies on the basic principles of protein- nucleic acid interactions that underlie the regulation of gene expression. During this reporting period we plan to focus on an analysis of the E. coli RNA polymerase transcription complex, and will use thermodynamic, kinetic and crosslinking methods to analyze the structure and function of the steady-state transcription elongation complex, the nature of pausing in this complex, and the relation of this pausing to """"""""factor-independent"""""""" and rho-dependent transcription termination. In these studies we will isolate and study specifically paused and stable lambdal and T7 phage elongation complexes. We will also use these complexes to examine the binding of various regulatory proteins (including sigma factor, nusA protein, rho protein, etc.) to the core polymerase of the elongation complex. In addition we will continue our studies of the mechanisms of transcript release by rho protein at rho-dependent transcript termination sites. Physico-chemical studies of the mechanisms of rho protein oligomerization, of RNA and ATP binding to rho. and of the RNA-dependent activation of rho ATPase will also be continued. Finally, we will continue to develop general theoretical and experimental methods for the study of protein-nucleic acid interactions. As before, we will attempt both to examine specific physiologically-active protein complexes involved in gene expression, and to elucidate some of the general principles that underlie the function of all such systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37GM015792-27
Application #
3484204
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-01-01
Project End
1997-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
27
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Schools of Arts and Sciences
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Johnson, Neil P; Ji, Huiying; Steinberg, Thomas H et al. (2015) Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6-Methyl Isoxanthopterin (6-MI) in DNA. J Phys Chem B 119:12798-807
Jose, Davis; Weitzel, Steven E; Baase, Walter A et al. (2015) Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity. Nucleic Acids Res 43:9291-305
Jose, Davis; Weitzel, Steven E; Baase, Walter A et al. (2015) Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding. Nucleic Acids Res 43:9276-90
Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos et al. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10:e0126420
Lee, Wonbae; von Hippel, Peter H; Marcus, Andrew H (2014) Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments. Nucleic Acids Res 42:5967-77
Phelps, Carey; Lee, Wonbae; Jose, Davis et al. (2013) Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions. Proc Natl Acad Sci U S A 110:17320-5
von Hippel, Peter H; Johnson, Neil P; Marcus, Andrew H (2013) Fifty years of DNA ""breathing"": Reflections on old and new approaches. Biopolymers 99:923-54
Lee, Wonbae; Jose, Davis; Phelps, Carey et al. (2013) A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex. Biochemistry 52:3157-70
Widom, Julia R; Rappoport, Dmitrij; Perdomo-Ortiz, Alejandro et al. (2013) Electronic transition moments of 6-methyl isoxanthopterin--a fluorescent analogue of the nucleic acid base guanine. Nucleic Acids Res 41:995-1004
Jose, Davis; Weitzel, Steven E; Jing, Debra et al. (2012) Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4. Proc Natl Acad Sci U S A 109:13596-601

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