A study of structure-function relationships in DNA-dependent RNA polymerase (RNAP) of Escherichia coli is proposed which will use a combination of protein-chemical and enzymological approaches. The ultimate goal of this study is the understanding of RNAP basic function and regulation in molecular detail, and the uncovering of the underlying structural determinants. The emphasis of this project is on topology of the ternary transcription complex as it propagates along DNA. We will test the model that the movement of the complex occurs in the """"""""inchworm"""""""" fashion whereby the enzyme compresses and extends with the amplitude of several nucleotides. Understanding of the mechanism of RNA polymerase propagation will provide clues to the design of this enzyme as a multifunctional molecular machine which serves as the target to factors that regulate gene expression. The proposed study will be focused on the RNA component of the ternary complex. Three principal approaches will be used. First we intend to use crosslinkable affinity probes incorporated into defined positions in the transcript to fix RNA in the complex, and then to study such complex enzymatically. The impediment to the normal RNA chain extension resulting from the crosslink will help understand the mechanism of RNAP propagation. The second approach employs the GreA protein, a novel transcript cleavage factor that we discovered, as a probe of RNA topology in the ternary complex. GreA cleaves the nascent transcript exposed in the vicinity of the enzyme's catalytic center. From the changes of the cleavage pattern that take place during complex propagation we expect to learn about the mechanism of the enzyme movement. Finally, we propose to identify amino acids involved in contacts with RNA in the ternary complex, and the length of DNA-RNA hybrid region by means of affinity labeling with probes incorporated into the transcript followed by mapping the adducts using methods of protein and nucleic acid chemistry.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM049242-01
Application #
3308590
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1993-06-01
Project End
1997-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Public Health Research Institute
Department
Type
DUNS #
City
Newark
State
NY
Country
United States
Zip Code
Mustaev, Arkadv; Zaychikov, Eugeny; Grachev, Mikhail et al. (2003) Strategies and methods of cross-linking of RNA polymerase active center. Methods Enzymol 371:191-206
Sosunov, Vasily; Sosunova, Ekaterina; Mustaev, Arkady et al. (2003) Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase. EMBO J 22:2234-44
Sosunova, Ekaterina; Sosunov, Vasily; Kozlov, Maxim et al. (2003) Donation of catalytic residues to RNA polymerase active center by transcription factor Gre. Proc Natl Acad Sci U S A 100:15469-74
Kazmierczak, K M; Davydova, E K; Mustaev, A A et al. (2002) The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases. EMBO J 21:5815-23
Kuznedelov, Konstantin; Korzheva, Nataliya; Mustaev, Arkady et al. (2002) Structure-based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA-DNA hybrid length. EMBO J 21:1369-78
Epshtein, Vitaliy; Mustaev, Arkady; Markovtsov, Vadim et al. (2002) Swing-gate model of nucleotide entry into the RNA polymerase active center. Mol Cell 10:623-34
Campbell, E A; Korzheva, N; Mustaev, A et al. (2001) Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell 104:901-12
Korzheva, N; Mustaev, A; Kozlov, M et al. (2000) A structural model of transcription elongation. Science 289:619-25
Godson, G N; Mustaev, A A; Sun, W (1998) ATP cross-linked to Escherichia coli single-strand DNA-binding protein can be utilized by the catalytic center of primase as initiating nucleotide for primer RNA synthesis on phage G4oric template. Biochemistry 37:3810-7
Nudler, E; Gusarov, I; Avetissova, E et al. (1998) Spatial organization of transcription elongation complex in Escherichia coli. Science 281:424-8

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