The long-term objective of this proposal is to probe deeper into the mechanism by which initiation of plasmid ColE1 DNA replication is controlled. The central molecular process in this control is the interaction of two plasmid-encoded RNA molecules. One RNA is the primer precursor transcript which is normally processed by RNase H to form a mature primer. Processing is negatively controlled by the interaction of the primer precursor with a small counterscript called RNA 1. RNA 1 is complementary to the 5'-terminal region of the primer precursor. RNA 1 is thought to alter the conformation of nascent primer RNA so that the primer is unable to form an RNA-DNA hybrid in the vicinity of the replication origin. A critical feature of the inhibition by RNA 1 is that it must interact with primer during a particular period of primer transcription. A major focus to work proposed here is the study of an unusual group of mutants that we have isolated which alter this interaction. These mutants show thermosensitive DNA amplification in vivo which results from single base changes in their primer RNA. The mutant primer is resistant to inhibition by wild type RNA 1 in vivo and in vitro. We believe that these mutants specifically alter the dynamic folding pathway of nascent primer RNA during transcription. We will test an RNA folding model we have proposed to explain the RNA 1 resistance of the mutant primers. Our folding model permits prediction of the replication phenotype of new mutants in the primer domain altered by the ts mutants. We will create these mutants to further test the model. We will study second-site revertants of these mutants that have wild type replication properties. We will also determine whether host mutants in which transcription components such as RNA polymerase and NusA are altered, suppress the amplification of the mutant plasmids in vivo. We will study the kinetics of plasmid DNA establishment in vivo by synchronously introducing plasmid into cells via plasmid infection. Plasmids are phage-plasmid hybrids; replication from the phage origin will be repressed by supplying phage repressor in trans. We are interested in the process by which plasmids amplify from a single copy to reach their steady-state levels. Again, we have specific mutations which are expected to alter plasmid establishment kinetics. These mutants alter specific GATC sequences in the primer promoter and consequently are not subject to an apparent replication block normally experienced by hemimethylated C01E1 DNA in vivo. We will monitor the expression of plasmid replication control elements such as RNA 1 and the Rop/Rorn protein during establishment of wild type and mutant plasmids in dam+ and dam-hosts and correlate this expression.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University Bloomington
Schools of Arts and Sciences
United States
Zip Code
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

Showing the most recent 10 out of 13 publications