E. Coli NusA Protein Binds in Vitro to an RNA Sequence Immediately Upstream of the BoxA Signal of Bacteriophage Lambda

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1985 Sep 1

PMID 2416564

Citations 7

Authors

A Tsugawa

T Kurihara

M Zuber

D L Court

Y Nakamura

Affiliations

Soon will be listed here.

Abstract

The NusA protein of Escherichia coli is a factor which mediates termination of transcription. In this paper, we demonstrate that the NusA protein can bind in vitro to a specific site on the mRNA of bacteriophage lambda. Several RNAs were synthesized by in vitro transcription of truncated lambda DNA templates, and the activity of NusA binding to these RNAs was examined by a Millipore filter-binding assay. RNAs containing the sequence immediately upstream of the boxA site were trapped on the filter by association with the NusA protein, but those lacking the site were not. Anti-NusA antibody inhibits this binding. To determine the binding site precisely, we developed a new method which we have named 'reverse-transcriptase mapping'. The RNA transcribed from the pL promoter was incubated with 32P-labelled DNA primer and NusA, and the primer-extension reaction was started by adding the reverse transcriptase. In this way, the primer extension was blocked at the position G of the boxA RNA sequence (5'CGCUCUUA 3'), indicating that the NusA-protection site is immediately upstream of boxA and includes the 5'-end C. The NusA protein purified from a temperature-sensitive nusA mutant defective in transcription termination showed reduced and thermolabile RNA-binding activity, suggesting that the RNA-binding activity is related to the physiological function of NusA.

Citing Articles

RNA-binding specificity of E. coli NusA.

Prasch S, Jurk M, Washburn R, Gottesman M, Wohrl B, Rosch P Nucleic Acids Res. 2009; 37(14):4736-42.

PMID: 19515940 PMC: 2724277. DOI: 10.1093/nar/gkp452.

Localization of nusA-suppressing amino acid substitutions in the conserved regions of the beta' subunit of Escherichia coli RNA polymerase.

Ito K, Nakamura Y Mol Gen Genet. 1996; 251(6):699-706.

PMID: 8757401 DOI: 10.1007/BF02174119.

Escherichia coli-Salmonella typhimurium hybrid nusA genes: identification of a short motif required for action of the lambda N transcription antitermination protein.

Craven M, GRANSTON A, Schauer A, Zheng C, Gray T, Friedman D J Bacteriol. 1994; 176(5):1394-404.

PMID: 8113180 PMC: 205205. DOI: 10.1128/jb.176.5.1394-1404.1994.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Chattopadhyay S, Garcia-Mena J, DeVito J, Wolska K, Das A Proc Natl Acad Sci U S A. 1995; 92(9):4061-5.

PMID: 7732031 PMC: 42102. DOI: 10.1073/pnas.92.9.4061.

Analysis of nutR, a site required for transcription antitermination in phage lambda.

Zuber M, Patterson T, Court D Proc Natl Acad Sci U S A. 1987; 84(13):4514-8.

PMID: 2955408 PMC: 305120. DOI: 10.1073/pnas.84.13.4514.

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