Skip to main content

Localization ofnusA-suppressing amino acid substitutions in the conserved regions of theβ′ subunit ofEscherichia coli RNA polymerase

Molecular and General Genetics MGG volume 251, pages 699–706 (1996)Cite this article

Abstract

Escherichia coli RNA polymerase is composed of four different subunits,α (present in two copies),β, β′ andσ. Among these, theβ′ polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood. We isolated novel mutations in a plasmidborne copy ofrpoC, which encodesβ′, as dominant suppressors of two temperature-sensitivenusA alleles. All 20 suppressors ofnusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in theβ′ subunit. In vivo and in vitro transcription assays revealed that therpoC10 allele ofβ′ participates in Rho-dependent transcription termination. On the other hand, of 20 suppressors ofnusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I. These results suggested that the conserved domains of theβ′ subunit ofE. coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • Allison LM, Moyle M, Shales M, Ingles C (1985) Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell 42:599–610

    Google Scholar 

  • Borukhov S, Lee J, Goldfarb A (1991) Mapping of a contact for the RNA 3′ termini in the largest subunit of RNA polymerase. J Biol Chem 266:23932–23935

    Google Scholar 

  • Burgess RR, Jendrisak JJ (1975) A procedure for the rapid, largescale purification ofEscherichia coli DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14:4634–4638

    Google Scholar 

  • Craven MG, Friedman DI (1991) Analysis of theEscherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes. J Bacteriol 173:1485–1491

    Google Scholar 

  • Farnham PJ, Greenblatt J, Platt T (1982) Effects of NusA protein on transcription termination in the tryptophan operon ofEscherichia coli. Cell 29:945–951

    Google Scholar 

  • Friedman DI, Gottesman M (1983) Lytic mode of lambda development. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA (eds) Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 21–51

    Google Scholar 

  • Friedman DI, Olson ER (1983) Evidence that a nucleotide sequence, “boxA”, is involved in the action of the NusA protein. Cell 34:143–149

    Google Scholar 

  • Friedman DI, Olson ER, Georgopoulos C, Tilly K, Herskowitz I, Banuett F (1984) Interactions of bacteriophage and host macromolecules in the growth of bacteriophage λ. Microbiol Rev 48:299–325

    Google Scholar 

  • Greenblatt J, Li J (1981a) Interaction of the sigma factor and thenusA gene protein ofE. coli with RNA polymerase in the initiation-termination cycle of transcription. Cell 24:421–428

    Google Scholar 

  • Greenblatt J, Li J (1981b) ThenusA gene product ofEscherichia coli: its identification and a demonstration that it interacts with geneN transcription antitermination protein of bacteriophage lambda. J Mol Biol 147:11–23

    Google Scholar 

  • Greenblatt J, McLimont M, Hanly S (1981) Termination of transcription bynusA gene protein ofEscherichia coli. Nature 292:215–220

    Google Scholar 

  • Gross C, Engbaek F, Flammang T, Burgess R (1976) Rapid micromethod for the purification ofEscherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis. J Bacteriol 128:382–389

    Google Scholar 

  • Horwitz RJ, Li V, Greenblatt J (1987) An elongation control particle containing theN gene transcriptional antitermination protein of bacteriophage lambda. Cell 51:631–641

    Google Scholar 

  • Ito K, Nakamura Y (1993) Pleiotropic effects of therpoC10 mutation affecting the RNA polymeraseβ′ subunit ofEscherichia coli on factor-dependent transcription termination and antitermination. Mol Microbiol 9:285–293

    Google Scholar 

  • Ito K, Egawa K, Nakamura Y (1991) Genetic interaction between theβ′ subunit of RNA polymerase and the arginine-rich domain ofEscherichia coli nusA protein. J Bacteriol 173:1492–1501

    Google Scholar 

  • Jin DJ, Gross CA (1989) ThreerpoBC mutations that suppress the termination defects ofrho mutants also affect the functions ofnusA mutants. Mol Gen Genet 216:269–275

    Google Scholar 

  • Jokerst RS, Weeks JR, Zehring WA, Greenleaf AL (1989) Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila. Mol Gen Genet 215:266–275

    Google Scholar 

  • Kassavetis GA, Chamberlin MJ (1981) Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. J Biol Chem 256:2777–2786

    Google Scholar 

  • Kawakami K, Inada T, Nakamura Y (1988) Conditionally lethal and recessive UGA-suppressor mutations in theprfB gene encoding peptide chain release factor 2 ofEscherichia coli. J Bacteriol 170:5378–5381

    Google Scholar 

  • Kingston RE, Chamberlin MJ (1981) Pausing and attenuation of in vitro transcription in therrnB operon ofE. coli. Cell 27:523–531

    Google Scholar 

  • Lau LF, Roberts JW (1985)ρ-dependent transcription termination at λtR1 requires upstream sequences. J Biol Chem 260:574–584

    Google Scholar 

  • Lau LF, Roberts JW, Wu R (1983) RNA polymerase pausing and transcript release at the λtR1 terminator in vitro. J Biol Chem 258:9391–9397

    Google Scholar 

  • Martinez E, Bartolomé B, de la Cruz F (1988) pACYC184-derived cloning vectors containing the multiple cloning site and lacZZ α reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68:159–162

    Google Scholar 

  • Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Mori A, Ito K, Mizobuchi K, Nakamura Y (1995) A transcription terminator signal necessary for plasmid ColIb-P9 replication. Mol Microbiol 17:291–301

    Google Scholar 

  • Nakamura Y (1978) RNA polymerase mutant with altered sigma factor inEscherichia coli. Mol Gen Genet 165:1–6

    Google Scholar 

  • Nakamura Y, Mizusawa S (1985) In vivo evidence that thenusA andinfB genes ofE. coli are part of the same multi-gene operon which encodes at least four proteins. EMBO J 4:527–532

    Google Scholar 

  • Nakamura Y, Mizusawa S, Court DL, Tsugawa A (1986a) Regulatory defects of a conditionally lethalnusAts mutant ofEscherichia coli; positive and negative modulator roles of NusA protein in vivo. J Mol Biol 189:103–111

    Google Scholar 

  • Nakamura Y, Mizusawa S, Tsugawa A, Imai M (1986b) Conditionally lethalnusAts mutation ofEscherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda. Mol Gen Genet 204:24–28

    Google Scholar 

  • Nakamura Y, Tsugawa A, Saito M, Egawa K (1987) Genetic dissection of thenusA protein ofEscherichia coli. In: Reznikoff WS, Burgess RR, Dahlberg JE, Gross CA, Record M, Wickens MP (eds) RNA polymerase and the regulation of transcription. Elsevier Press, New York, pp 367–380

    Google Scholar 

  • Robledo R, Atkinson BL, Gottesman ME (1991)Escherichia coli mutations that block transcription termination by phage HK022 Nun protein. J Mol Biol 220:613–619

    Google Scholar 

  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Schmidt MC, Chamberlin MJ (1984) Binding ofrho factor toEscherichia coli RNA polymerase mediated bynusA protein. J Biol Chem 259:15000–15002

    Google Scholar 

  • Tsugawa A, Kurihara T, Zuber M, Court DL, Nakamura Y (1985)E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of theboxA signal of bacteriophage lambda. EMBO J 4:2337–2342

    Google Scholar 

  • Tsugawa A, Saito M, Court DL, Nakamura Y (1988)nusA amber mutations that cause temperature-sensitive growth ofEscherichia coli. J Bacteriol 170:908–915

    Google Scholar 

  • Tsurushita N, Shigesada K, Imai M (1989) Mutant rho factors with increased transcription termination activities. I. Functional correlations of the primary and secondary polynucleotide binding sites with the efficiency and site-selectivity of rho-dependent termination. J Mol Biol 210:23–27

    Google Scholar 

  • Weilbaecher R, Hebron C, Feng G, Landick R (1994) Termination-altering amino acid substitutions in theβ′ subunit ofEscherichia coli RNA polymerase identify regions involved in RNA chain elongation. Genes Dev 8:2913–2927

    Google Scholar 

  • Young RA (1991) RNA polymerase II. Annu Rev Biochem 60:689–715

    Google Scholar 

Download references

Author information

Affiliations

  1. Department of Tumor Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108, Tokyo, Japan

    K. Ito & Y. Nakamura

Authors
  1. K. Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y. Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by M. Sekiguchi

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ito, K., Nakamura, Y. Localization ofnusA-suppressing amino acid substitutions in the conserved regions of theβ′ subunit ofEscherichia coli RNA polymerase. Molec. Gen. Genet. 251, 699–706 (1996). https://doi.org/10.1007/BF02174119

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02174119

Key words

  • RNA polymerase
  • β′ subunit
  • NusA
  • Transcription termination
  • Rho factor