# MUTATIONAL ANALYSIS OF E. COLI CORE RNA POLYMERASE

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 1990 · $212,299

## Abstract

Our long term goal is to understand the structural and functional
organization of E. coli RNA polymerase.  Such knowledge is crucial
to understanding how RNA polymerase performs elementary steps in
transcription and prerequisite for a molecular understanding of how
small molecule and protein factors regulate RNA polymerase
activity.  The selection and analysis of mutations is an excellent
point of entry to this problem.  Intensive analysis of particular
mutant classes defines limited areas of the protein, and
characterization of the in vivo phenotypes suggests the likely
functional alterations.  These results can then be used to design
experiments to determine if the mutational defect affects
transcription in vitro in the way suggested by in vivo analysis and
to demonstrate directly that a particular region of protein
carriers out the proposed function.  Thus our specific aims are:

1. The role of the sigma subunit in determining promoter
specificity.

We will analyze the effects of sigma 70 mutants on kinetics of
promoter recognition select mutations in sigma 32 with altered
promoter recognition, and make sigma 70-sigma 32 hybrids to
localize the region of sigma interacting with the -10 region of the
promoter.  We will determine if intact sigma 70 and sigma 32 and
portions of each of these sigma's bind to promoter DNA.  If
successful, we will design a partial sigma that acts as DNA binding
molecule in vivo, for structural and mutational analysis.

2.  To analyze the molecular basis for the pleiotropic effects of
Rifr mutations.
We will determine whether selected Rifr mutations affect various
steps in RNA synthesis which can be monitored in vitro, including:
formation of the DNA/RNA hybrid region in the transcription bubble,
RNA affinity to the beta subunit, K/M's for nucleotide binding,
abortive initiation and pausing; create an antibody population
which is specific for the major Rif domain to enable us to monitor
whether this region is involved in the binding of rifampicin,
substrate or nascent RNA and map and characterize allele specific
second site revertants of Rifr mutations to probe for regions of
beta and beta' which lie in close proximity to the Rif domain.

3.  To obtain mutations that affect specific functions of RNA We
will improve the genetic technology for selecting RNA polymerase
mutations, and, by selecting, mapping and analyzing mutations,
locate regions of RNA polymerase involved some of the following
functions:  nucleotide binding, translocation, NusA binding, and
termination.

## Key facts

- **NIH application ID:** 3128976
- **Project number:** 5R01AI019635-08
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **SAM.gov UEI:** LCLSJAGTNZQ7
- **Principal Investigator:** CAROL Anne GROSS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1990
- **Award amount:** $212,299
- **Award type:** 5
- **Project period:** 1983-01-01 → 1992-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3128976

## Citation

> US National Institutes of Health, RePORTER application 3128976, MUTATIONAL ANALYSIS OF E. COLI CORE RNA POLYMERASE (5R01AI019635-08). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3128976. Licensed CC0.

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