# MUTATIONAL ANALYSIS OF E COLI CORE RNA POLYMERASE

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 1993 · $247,421

## Abstract

Our fundamental interest is a central biological problem -- how does the
cell regulate transfer of information from gene to protein?  We focus on
RNA polymerase, the enzyme responsible for transcription.  We view this
enzyme as the ultimate target of a regulatory system that adjusts
transcription in response to intracellular and extracellular signals by
modulating the enzymatic functions of RNA polymerase.  To understand this
regulatory system, we first identify regions of RNA polymerase involved
in a particular function by selecting, mapping and sequencing particular
classes of mutants, or where appropriate by crosslinking.  We then
investigate how altering individual functions affects gene regulation.
Because the subunit structure of RNA polymerase is highly conserved, our
structure-function analysis, which is most feasible in E. coli, should
have broad applicability.  In the current granting period we will:

1.   Further define the role of sigma in transcription initiation

     We will select mutations in sigma32 that block initiation in vivo
     in the presence of the wild type sigma32 and determine whether the
     mutants are defective in strand opening, phosphodiester bond
     formation or sigma release.  In addition, we will further delineate
     the promoter recognition properties of sigma by determining which
     mutational changes in promoter DNA affect recognition by fragments
     of sigma70 and assessing the effects of alanine substitution
     mutations in sigma32 on promoter recognition.

2.   Define the interactions between sigma and core RNA polymerase

     We will select point mutants in sigma32 and sigma70 that are
     defective in binding to core RNA polymerase and use them to obtain
     compensatory mutations in core RNA polymerase.  Mutants will be
     characterized for their functional and regulatory effects.

3.   Define functional domains in core RNA polymerase

     a)   interaction with NusA.  To define the site(s) on polymerase
          that bind NusA and allow us to further determine the role(s)
          of NusA in vivo, we will screen a group of RNA polymerase
          mutants defective in NusA dependent termination for those
          defective in binding or responding to NusA.

     b)   substrate binding and translocation.  To further characterize
          the regions that comprise the active site of RNA polymerase,
          we will identify and characterize in vitro mutants that are
          resistant to streptolydigin and mutants that elongate more
          slowly than wild-type.

     c)   binding to DNA.  We will use chemical crosslinking to identify
          regions of core RNA polymerase close to DNA during initiation,
          transition to elongation and elongation.

## Key facts

- **NIH application ID:** 3128971
- **Project number:** 2R01AI019635-11
- **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:** 1993
- **Award amount:** $247,421
- **Award type:** 2
- **Project period:** 1983-01-01 → 1993-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3128971

## Citation

> US National Institutes of Health, RePORTER application 3128971, MUTATIONAL ANALYSIS OF E COLI CORE RNA POLYMERASE (2R01AI019635-11). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3128971. Licensed CC0.

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