Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Sep;4(9):2337–2342. doi: 10.1002/j.1460-2075.1985.tb03935.x

E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda.

A Tsugawa, T Kurihara, M Zuber, D L Court, Y Nakamura
PMCID: PMC554506  PMID: 2416564

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.

Full text

PDF
2337

Images in this article

2340Fig. 6.
on p.2340
2341Fig. 7.
on p.2341

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Farnham P. J., Greenblatt J., Platt T. Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli. Cell. 1982 Jul;29(3):945–951. doi: 10.1016/0092-8674(82)90457-3. [DOI] [PubMed] [Google Scholar]
  2. Franklin N. C., Bennett G. N. The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic. Gene. 1979 Dec;8(1):107–119. doi: 10.1016/0378-1119(79)90011-8. [DOI] [PubMed] [Google Scholar]
  3. Friedman D. I., Olson E. R. Evidence that a nucleotide sequence, "boxA," is involved in the action of the NusA protein. Cell. 1983 Aug;34(1):143–149. doi: 10.1016/0092-8674(83)90144-7. [DOI] [PubMed] [Google Scholar]
  4. Greenblatt J., McLimont M., Hanly S. Termination of transcription by nusA gene protein of Escherichia coli. Nature. 1981 Jul 16;292(5820):215–220. doi: 10.1038/292215a0. [DOI] [PubMed] [Google Scholar]
  5. Gross C., Engbaek F., Flammang T., Burgess R. Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis. J Bacteriol. 1976 Oct;128(1):382–389. doi: 10.1128/jb.128.1.382-389.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ishii S., Ihara M., Maekawa T., Nakamura Y., Uchida H., Imamoto F. The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli. Nucleic Acids Res. 1984 Apr 11;12(7):3333–3342. doi: 10.1093/nar/12.7.3333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kassavetis G. A., Chamberlin M. J. Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. J Biol Chem. 1981 Mar 25;256(6):2777–2786. [PubMed] [Google Scholar]
  8. Kingston R. E., Chamberlin M. J. Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli. Cell. 1981 Dec;27(3 Pt 2):523–531. doi: 10.1016/0092-8674(81)90394-9. [DOI] [PubMed] [Google Scholar]
  9. Kung H., Spears C., Weissbach H. Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase. J Biol Chem. 1975 Feb 25;250(4):1556–1562. [PubMed] [Google Scholar]
  10. Kurihara T., Nakamura Y. Cloning of the nusA gene of Escherichia coli. Mol Gen Genet. 1983;190(2):189–195. doi: 10.1007/BF00330639. [DOI] [PubMed] [Google Scholar]
  11. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  12. Musso R. E., Di Lauro R., Adhya S., de Crombrugghe B. Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell. 1977 Nov;12(3):847–854. doi: 10.1016/0092-8674(77)90283-5. [DOI] [PubMed] [Google Scholar]
  13. Nakamura Y. Hybrid plasmid carrying Escherichia coli genes for the primase (dnaG) and RNA polymerase sigma factor (rpoD); gene organization and control of their expression. Mol Gen Genet. 1980;178(3):487–497. doi: 10.1007/BF00337853. [DOI] [PubMed] [Google Scholar]
  14. Nakamura Y., Mizusawa S. In vivo evidence that the nusA and infB genes of E. coli are part of the same multi-gene operon which encodes at least four proteins. EMBO J. 1985 Feb;4(2):527–532. doi: 10.1002/j.1460-2075.1985.tb03660.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nakamura Y., Uchida H. Isolation of conditionally lethal amber mutations affecting synthesis of the nusA protein of Escherichia coli. Mol Gen Genet. 1983;190(2):196–203. doi: 10.1007/BF00330640. [DOI] [PubMed] [Google Scholar]
  16. Olson E. R., Flamm E. L., Friedman D. I. Analysis of nutR: a region of phage lambda required for antitermination of transcription. Cell. 1982 Nov;31(1):61–70. doi: 10.1016/0092-8674(82)90405-6. [DOI] [PubMed] [Google Scholar]
  17. Salstrom J. S., Szybalski W. Coliphage lambdanutL-: a unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription. J Mol Biol. 1978 Sep 5;124(1):195–221. doi: 10.1016/0022-2836(78)90156-0. [DOI] [PubMed] [Google Scholar]
  18. Shigesada K., Imai M. Studies on the altered rho factor in nitA mutants of Escherichia coli defective in transcription termination. II. Purification and molecular properties of the mutant rho. J Mol Biol. 1978 Apr 25;120(4):467–486. doi: 10.1016/0022-2836(78)90349-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES