Abstract
The DNA sequence of the secA gene, essential for protein export in Escherichia coli, was determined and found to encode a hydrophilic protein of 901 amino acid residues with a predicted molecular weight of 101,902, consistent with its previously determined size and subcellular location. Sequence analysis of 9 secA(Ts) mutations conferring general protein export and secA regulatory defects revealed that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein and were the result of single amino acid changes predicted to be severely disruptive of protein structure and function. The DNA sequence immediately upstream of secA was shown to encode a previously inferred gene, gene X. Sequence analysis of a conditionally lethal amber mutation, am109, previously inferred to be located proximally in the secA gene, revealed that it was located distally in gene X and was conditionally lethal due to its polar effect on secA expression. This and additional evidence are presented indicating that gene X and secA are cotranscribed.
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- Alton N. K., Vapnek D. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature. 1979 Dec 20;282(5741):864–869. doi: 10.1038/282864a0. [DOI] [PubMed] [Google Scholar]
- Anderson C. W., Lewis J. B. Amino-terminal sequence of adenovirus type 2 proteins: hexon, fiber, component IX, and early protein 1B-15K. Virology. 1980 Jul 15;104(1):27–41. doi: 10.1016/0042-6822(80)90363-3. [DOI] [PubMed] [Google Scholar]
- Bankaitis V. A., Bassford P. J., Jr Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope. J Bacteriol. 1985 Jan;161(1):169–178. doi: 10.1128/jb.161.1.169-178.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beall B., Lutkenhaus J. Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli. J Bacteriol. 1987 Dec;169(12):5408–5415. doi: 10.1128/jb.169.12.5408-5415.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cerretti D. P., Dean D., Davis G. R., Bedwell D. M., Nomura M. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene. Nucleic Acids Res. 1983 May 11;11(9):2599–2616. doi: 10.1093/nar/11.9.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen L., Tai P. C. ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384–4388. doi: 10.1073/pnas.82.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen P., Duewer T., Fischer E. H. Phosphorylase from dogfish skeletal muscle. Purification and a comparison of its physical properties to those of rabbit muscle phosphorylase. Biochemistry. 1971 Jul 6;10(14):2683–2694. doi: 10.1021/bi00790a005. [DOI] [PubMed] [Google Scholar]
- Emr S. D., Hanley-Way S., Silhavy T. J. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. doi: 10.1016/0092-8674(81)90272-5. [DOI] [PubMed] [Google Scholar]
- Evans E. A., Gilmore R., Blobel G. Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci U S A. 1986 Feb;83(3):581–585. doi: 10.1073/pnas.83.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gardel C., Benson S., Hunt J., Michaelis S., Beckwith J. secD, a new gene involved in protein export in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1286–1290. doi: 10.1128/jb.169.3.1286-1290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
- Geller B. L., Movva N. R., Wickner W. Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4219–4222. doi: 10.1073/pnas.83.12.4219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
- Inouye H., Barnes W., Beckwith J. Signal sequence of alkaline phosphatase of Escherichia coli. J Bacteriol. 1982 Feb;149(2):434–439. doi: 10.1128/jb.149.2.434-439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito K., Bassford P. J., Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. doi: 10.1016/0092-8674(81)90097-0. [DOI] [PubMed] [Google Scholar]
- Ito K. Identification of the secY (prlA) gene product involved in protein export in Escherichia coli. Mol Gen Genet. 1984;197(2):204–208. doi: 10.1007/BF00330964. [DOI] [PubMed] [Google Scholar]
- Jones C. A., Holland I. B. Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli. EMBO J. 1984 May;3(5):1181–1186. doi: 10.1002/j.1460-2075.1984.tb01948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumamoto C. A., Beckwith J. Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol. 1985 Jul;163(1):267–274. doi: 10.1128/jb.163.1.267-274.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumamoto C. A., Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. doi: 10.1128/jb.154.1.253-260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
- Liss L. R., Oliver D. B. Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins. J Biol Chem. 1986 Feb 15;261(5):2299–2303. [PubMed] [Google Scholar]
- Lutkenhaus J. F., Wolf-Watz H., Donachie W. D. Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus (ftsZ). J Bacteriol. 1980 May;142(2):615–620. doi: 10.1128/jb.142.2.615-620.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Müller M., Blobel G. Protein export in Escherichia coli requires a soluble activity. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7737–7741. doi: 10.1073/pnas.81.24.7737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
- Oliver D. B., Beckwith J. Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli. J Bacteriol. 1982 May;150(2):686–691. doi: 10.1128/jb.150.2.686-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver D. B., Beckwith J. Regulation of a membrane component required for protein secretion in Escherichia coli. Cell. 1982 Aug;30(1):311–319. doi: 10.1016/0092-8674(82)90037-x. [DOI] [PubMed] [Google Scholar]
- Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
- Rollo E. E., Oliver D. B. Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants. J Bacteriol. 1988 Jul;170(7):3281–3282. doi: 10.1128/jb.170.7.3281-3282.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schekman R. Protein localization and membrane traffic in yeast. Annu Rev Cell Biol. 1985;1:115–143. doi: 10.1146/annurev.cb.01.110185.000555. [DOI] [PubMed] [Google Scholar]
- Schreier P. H., Cortese R. A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13. J Mol Biol. 1979 Mar 25;129(1):169–172. doi: 10.1016/0022-2836(79)90068-8. [DOI] [PubMed] [Google Scholar]
- Seery V. L., Fischer E. H., Teller D. C. Subunit structure of glycogen phosphorylase. Biochemistry. 1970 Sep 1;9(18):3591–3598. doi: 10.1021/bi00820a014. [DOI] [PubMed] [Google Scholar]
- Semler B. L., Anderson C. W., Kitamura N., Rothberg P. G., Wishart W. L., Wimmer E. Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3464–3468. doi: 10.1073/pnas.78.6.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shiba K., Ito K., Yura T., Cerretti D. P. A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J. 1984 Mar;3(3):631–635. doi: 10.1002/j.1460-2075.1984.tb01859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sullivan N. F., Donachie W. D. Transcriptional organization within an Escherichia coli cell division gene cluster: direction of transcription of the cell separation gene envA. J Bacteriol. 1984 Nov;160(2):724–732. doi: 10.1128/jb.160.2.724-732.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Titani K., Koide A., Hermann J., Ericsson L. H., Kumar S., Wade R. D., Walsh K. A., Neurath H., Fischer E. H. Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4762–4766. doi: 10.1073/pnas.74.11.4762. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokunaga M., Loranger J. M., Wu H. C. Prolipoprotein modification and processing enzymes in Escherichia coli. J Biol Chem. 1984 Mar 25;259(6):3825–3830. [PubMed] [Google Scholar]
- Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
- Watts C., Wickner W., Zimmermann R. M13 procoat and a pre-immunoglobulin share processing specificity but use different membrane receptor mechanisms. Proc Natl Acad Sci U S A. 1983 May;80(10):2809–2813. doi: 10.1073/pnas.80.10.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
- Wiedmann M., Kurzchalia T. V., Hartmann E., Rapoport T. A. A signal sequence receptor in the endoplasmic reticulum membrane. 1987 Aug 27-Sep 2Nature. 328(6133):830–833. doi: 10.1038/328830a0. [DOI] [PubMed] [Google Scholar]
- Wolfe P. B., Rice M., Wickner W. Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli. J Biol Chem. 1985 Feb 10;260(3):1836–1841. [PubMed] [Google Scholar]
- Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]