The long-term aim of the experiments proposed here is a comprehensive description of the molecular rules governing the frequency of initiation of DNA replication in a stably inherited replicon. Our model system for these experiments is the E. coli plasmid ColE1 and its derivatives. Experiments are described which analyze the three plasmid-encoded elements known to play central roles in replication control of ColE1: the RNA primer for replication; a small RNA, designated RNA1, that negatively modulates the frequency of processing of the primer by a unique RNA-RNA interaction; and the rop gene product, a small polypeptide that acts as a second negative control element on primer formation. A central tool for these experiments is the isolation of mutants in these control elements that affect plasmid copy number and/or incompatibility properties. Procedures are described to isolate mutants in each of the control elements and to produce these altered elements in large quantities. The interaction between RNA1 and primer precursor is extremely sensitive to perturbations in the RNA secondary and tertiary structures of these molecules. A major aim of this proposal is to define the structural domains of wild-type primer RNA and compare these domains to those displayed by specific primer mutants. Methods are described for the isolation and characterization of RNA1 mutants and for analysis of RNA1-primer interaction in vitro. Experiments are proposed to investigate the mechanisms by which the rop polypeptide affects primer formation. Methods for isolation of rop structural mutations and mutations in the rop-interaction site are proposed. Efforts will be made to isolate a rop-RNA1 ribonucleoprotein complex which may play a role in primer interaction. The physical interaction between mutant rop and mutant RNA1 species will also be explored, as will the structural consequnces for primer RNA when produced in the presence of rop-RNA1. Finally the roles of specific host genes in modulating the synthesis or activity of the ColE1 control elements will be explored. We will pursue the possibility that the E. coli dam methylase is involved in rop function and that the dnaA gene is a negative control element for synthesis of the RNA1 gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024212-11
Application #
3272117
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
11
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Indiana University Bloomington
Department
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47402
Yang, Y L; Polisky, B (1999) Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli. Plasmid 41:55-62
Yang, Y L; Polisky, B (1993) Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli. J Bacteriol 175:428-37
Merlin, S; Polisky, B (1993) Analysis of establishment phase replication of the plasmid ColE1. J Mol Biol 230:137-50
Fitzwater, T; Yang, Y L; Zhang, X Y et al. (1992) Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. J Mol Biol 226:997-1008
Patnaik, P K; Merlin, S; Polisky, B (1990) Effect of altering GATC sequences in the plasmid ColE1 primer promoter. J Bacteriol 172:1762-8
Polisky, B; Zhang, X Y; Fitzwater, T (1990) Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants. EMBO J 9:295-304
Gilley, D; Preer Jr, J R; Aufderheide, K J et al. (1988) Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol Cell Biol 8:4765-72
Dooley, T P; Polisky, B (1987) Suppression of ColE1 RNA-RNA mismatch mutations in vivo by the ColE1 Rop protein. Plasmid 18:24-34
Godiska, R; Aufderheide, K J; Gilley, D et al. (1987) Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci U S A 84:7590-4
Polisky, B; Tamm, J; Fitzwater, T (1985) Construction of Co1E1 RNA1 mutants and analysis of their function in vivo. Basic Life Sci 30:321-33

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