# NOVEL TRANSCRIBING ACTIVITIES IN N-4 INFECTED E COLI

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 1992 · $190,026

## Abstract

We will use biochemical and genetic approaches to analyze the
mechanism of promoter utilization by the three DNA-dependent RNA
polymerases required during bacteriophage N4 development.

Early N4 RNAs are transcribed by a virion-encapsulated, phage-
coded, rifampicin-resistant RNA polymerase (vRNAP) which recognizes
its promoters on single-stranded DNA with in vivo specificity.
Promoter recognition in vivo requires supercoiled template and E.
coli single-stranded DNA binding protein (SSB).  The role of
supercoiling and SSB in activation of N4 vRNAP promoters will be
determined by footprinting, affinity chromatography and the
isolation of SSB mutants specifically altered in N4 early RNA
synthesis.  The gene for the vRNAP will be cloned in order to
eventually manipulate it to determine functional domains in the
protein.  The fate of the vRNAP after injection will be determined.

Middle N4 RNAs are synthesized by a second phage-coded, rifampicin-
resistant RNA polymerase (N4 RNAP II) composed of two polypeptides
30,000 and 40,000 MW which, in vitro, does not utilize double-
stranded DNA.  A third polypeptide, 15,000 MW, is required in vivo.
Recently, we have determined the in vivo sites of transcription
initiation and developed an in vitro system which utilizes these
sites in vitro and is dependent on the activity of the 15,000,
30,000 and 40,000 MW polypeptides.  We will determine the relevance
of the conserved sequences at the sites of transcription initiation
to promoter activity by deletion and mutation analysis.  The 15,000
MW polypeptide will be purified, and its role in N4 RNAP II
promoter recognition studied.  The N4 RNAP II polypeptides will be
cloned in order to initiate structural studies.

Late N4 transcription requires the activity of the E. coli RNA
polymerase.  Sites of late transcription initiation do not show
homology at -35 and -10, a conserved octanucleotide is located just
upstream of +1.  These sites are poor templates for E. coli RNA
polymerase when present on linear DNA.  They are activated upon
template supercoiling.  Recent results suggest that the N4-coded
single-stranded DNA binding protein (DBP) is required.  The kinetic
parameters of N4 late promoters will be determined to gain insights
into the role of supercoiling in promoter activation.  The
interaction of the N4 DBP with late promoters will be studied.  We
will attempt to isolate mutants in the N4 DBP which specifically
affect N4 late transcription.

## Key facts

- **NIH application ID:** 3125230
- **Project number:** 5R01AI012575-18
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **SAM.gov UEI:** ZUE9HKT2CLC9
- **Principal Investigator:** LUCIA B. B ROTHMAN-DENES
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1992
- **Award amount:** $190,026
- **Award type:** 5
- **Project period:** 1988-05-01 → 1993-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3125230

## Citation

> US National Institutes of Health, RePORTER application 3125230, NOVEL TRANSCRIBING ACTIVITIES IN N-4 INFECTED E COLI (5R01AI012575-18). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3125230. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*