# STRUCTURE AND GENETIC CONTROL OF COLICINES

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 1987 · $186,563

## Abstract

This proposal is concerned with a genetic and biochemical analysis of the
regulation of initiation of replication of the antibiotic resistance
plasmid R6K in Escherichia coli.  This multi-copy plasmid is 38 kilobases
in size and specifies resistance to the antibiotics ampicillin and
streptomycin.  Previous studies have defined a contiguous replication
region that spans 4 kilobases in size.  Within this region three origins of
replication, designated Alpha, Beta and Gamma, have been identified and
shown to function in vivo and in an in vitro replication system.  In
addition, a gene (pir) specifies a replication initiation protein (Pi) is
located between the Gamma and Beta origins and is required for activity of
all three origins.  The Pi protein is multi-functional in that it exhibits
positive and negative activity in the initiation of R6K replication and
autoregulates the expression of the pir gene.  The activity of the three
replication origins also requires in cis seven 22 base pair direct repeats
located in the Gamma-origin region.  Molecular genetic and biochemical
analysis will be directed at determining the role of the Pi protein in the
regulation of initiation of plasmid R6K with emphasis on the nature of the
interaction of the Pi protein with the direct repeat region, other segments
of the R6K replicon, and E. coli replication proteins.  The introduction of
wild-type and mutant forms of the Pi protein and the direct repeats will be
analyzed using gel electrophoresis, electron microscopy and NMR
spectroscopy techniques.  The effect of mutational changes in the direct
repeat region and the Pi protein on the formation of RNA transcripts of the
R6K replicons and replication also will be examined in vitro.  An R6K gene
product that directs the initiation of replication from the Beta-origin has
been identified and will be characterized.  In addition molecular genetic
approaches will be directed at the mechanism of autoregulation of pir gene
expression and the role of this autoregulation in plasmid copy number
control.  Finally, the role of E. coli host proteins in R6K replication
will be explored by isolating and characterizing E. coli mutants that alter
the replication properties of wild-type and mutant R6K plasmids.  These
experimental approaches are designed to elucidate the major components of
the regulatory machinery and the nature of their interactions responsible
for the control of the copy number of plasmid R6K, a member of a major
group of plasmids characterized by the presence of direct repeats at a
replication origin and a plasmid encoded replication protein.

## Key facts

- **NIH application ID:** 3124347
- **Project number:** 5R01AI007194-22
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **SAM.gov UEI:** UYTTZT6G9DT1
- **Principal Investigator:** DONALD R HELINSKI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1987
- **Award amount:** $186,563
- **Award type:** 5
- **Project period:** 1978-02-01 → 1991-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3124347

## Citation

> US National Institutes of Health, RePORTER application 3124347, STRUCTURE AND GENETIC CONTROL OF COLICINES (5R01AI007194-22). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3124347. Licensed CC0.

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