In this work we will exploit the preliminary observation that non-denaturing polyacrylamide gel electrophoresis is able to separate discrete intermediates in the pathway of transcription initiation. These have been tentatively identified as """"""""closed"""""""" and """"""""open"""""""" polymerase-promoter complexes, and an """"""""initiated"""""""" complex containing a stably bound nascent RNA. The objectives include 1) further confirmation of the complex identities, 2) characterization of equilibria and kinetics for conversion among the states, and 3) use of enzymatic and chemical footprinting methods to identify the protein-DNA contact regions. A new method, called functional domain mapping, will be used to discover what region of the DNA sequence is required for formation of a particular polymerase-promoter complex which is stable enough to withstand gel electrophoresis. In addition, 4) we will search for DNA conformational modification effects, such as systematic bending or increased flexibility, in the discrete initiation intermediates, 5) examine the complexes for protein structural changes, such as loss of particular subunits, (especially sigma), and changes in protein-protein contacts as revealed by chemical crosslinking, and 6) examine ternary complexes formed in the presence of lac repressor or the cAMP-binding gene activating protein CAP. These latter experiments will seek to elucidate the effect of gene regulatory proteins on the steps of transcription initiation, through study of their influence on kinetics and equilibria of the reaction steps, and on conformational influences such as DNA bending. Protein-protein contacts in discrete ternary complexes will be investigated by chemical crosslinking methods. Finally, 7) we will study the rate and mechanism of conversion from a complex undergoing abortive initiation to the complex with stably bound RNA. Additional aims shared with other projects include study of the effect of DNA-binding antitumor drugs on the stages and intermediates in transcription initiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034205-02
Application #
3284778
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-12-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Gartenberg, M R; Crothers, D M (1991) Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter. J Mol Biol 219:217-30
Zinkel, S S; Crothers, D M (1991) Catabolite activator protein-induced DNA bending in transcription initiation. J Mol Biol 219:201-15
Gartenberg, M R; Ampe, C; Steitz, T A et al. (1990) Molecular characterization of the GCN4-DNA complex. Proc Natl Acad Sci U S A 87:6034-8
Straney, D C; Straney, S B; Crothers, D M (1989) Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J Mol Biol 206:41-57
Brown, A M; Crothers, D M (1989) Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP. Proc Natl Acad Sci U S A 86:7387-91
Ghosaini, L R; Brown, A M; Sturtevant, J M (1988) Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry 27:5257-61
Straney, S B; Crothers, D M (1987) Lac repressor is a transient gene-activating protein. Cell 51:699-707
Straney, D C; Crothers, D M (1987) A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J Mol Biol 193:267-78
Straney, D C; Crothers, D M (1987) Effect of drug-DNA interactions upon transcription initiation at the lac promoter. Biochemistry 26:1987-95
Straney, D C; Crothers, D M (1987) Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter. J Mol Biol 193:279-92

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