Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Feb;173(4):1478–1484. doi: 10.1128/jb.173.4.1478-1484.1991

Effects of translation on degradation of mRNA segments transcribed from the polycistronic puf operon of Rhodobacter capsulatus.

G Klug 1, S N Cohen 1
PMCID: PMC207285  PMID: 1995592

Abstract

Previous work has shown that expression of genes within the polycistronic puf operon of Rhodobacter capsulatus is regulated in part by differential degradation of segments of puf transcripts. To understand the role of ribosome coverage in the differential stability of puf mRNA segments, we have studied the effects of mutations that alter translation of specific puf transcript segments on puf mRNA decay. Our results show that stopping translation either within the light-harvesting I (LHI) genes or near the 5' end of the reaction center (RC)-coding region decreased the stability of puf transcript segments downstream from a hairpin loop structure located between the LHI and RC genes but failed to affect the upstream sequences so long as the loop was present. Mutations that allowed translation to proceed through the hairpin structure reduced its ability to protect upstream sequences from accelerated decay. Introduction of translation stops more than 107 bp into the RC-coding region, but still 5' to an mRNA segment containing decay-promoting endonuclease cleavage sites, had no effect on puf mRNA stability. The divergent and location-dependent consequences of translation stops imply that different mechanisms are responsible for the degradation of different puf mRNA segments and indicate that coverage of puf mRNA sequences by ribosomes is insufficient and may in some cases be unnecessary to protect these sequences from degradation.

Full text

PDF
1478

Images in this article

1480Image
on p.1480
1480Image
on p.1480
1481Image
on p.1481
1482Image
on p.1482
1482Image
on p.1482

Selected References

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

  1. Adams C. W., Forrest M. E., Cohen S. N., Beatty J. T. Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon. J Bacteriol. 1989 Jan;171(1):473–482. doi: 10.1128/jb.171.1.473-482.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer C. E., Marrs B. L. Rhodobacter capsulatus puf operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7074–7078. doi: 10.1073/pnas.85.19.7074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauer C. E., Young D. A., Marrs B. L. Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene. J Biol Chem. 1988 Apr 5;263(10):4820–4827. [PubMed] [Google Scholar]
  4. Beck E., Sommer R., Auerswald E. A., Kurz C., Zink B., Osterburg G., Schaller H., Sugimoto K., Sugisaki H., Okamoto T. Nucleotide sequence of bacteriophage fd DNA. Nucleic Acids Res. 1978 Dec;5(12):4495–4503. doi: 10.1093/nar/5.12.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belasco J. G., Beatty J. T., Adams C. W., von Gabain A., Cohen S. N. Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript. Cell. 1985 Jan;40(1):171–181. doi: 10.1016/0092-8674(85)90320-4. [DOI] [PubMed] [Google Scholar]
  6. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  7. Båga M., Göransson M., Normark S., Uhlin B. E. Processed mRNA with differential stability in the regulation of E. coli pilin gene expression. Cell. 1988 Jan 29;52(2):197–206. doi: 10.1016/0092-8674(88)90508-9. [DOI] [PubMed] [Google Scholar]
  8. Chen C. Y., Beatty J. T., Cohen S. N., Belasco J. G. An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability. Cell. 1988 Feb 26;52(4):609–619. doi: 10.1016/0092-8674(88)90473-4. [DOI] [PubMed] [Google Scholar]
  9. Chen C. Y., Belasco J. G. Degradation of pufLMX mRNA in Rhodobacter capsulatus is initiated by nonrandom endonucleolytic cleavage. J Bacteriol. 1990 Aug;172(8):4578–4586. doi: 10.1128/jb.172.8.4578-4586.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Imamoto F., Kano Y. Inhibition of transcription of the tryptophan operon in Escherichia coli by a block in initiation of translation. Nat New Biol. 1971 Aug 11;232(2):169–173. doi: 10.1038/newbio232169a0. [DOI] [PubMed] [Google Scholar]
  12. Kennell D., Simmons C. Synthesis and decay of messenger ribonucleic acid from the lactose operon of Escherichia coli during amino-acid starvation. J Mol Biol. 1972 Oct 14;70(3):451–464. doi: 10.1016/0022-2836(72)90552-9. [DOI] [PubMed] [Google Scholar]
  13. Klug G., Adams C. W., Belasco J., Doerge B., Cohen S. N. Biological consequences of segmental alterations in mRNA stability: effects of deletion of the intercistronic hairpin loop region of the Rhodobacter capsulatus puf operon. EMBO J. 1987 Nov;6(11):3515–3520. doi: 10.1002/j.1460-2075.1987.tb02677.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klug G., Cohen S. N. Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA. J Bacteriol. 1990 Sep;172(9):5140–5146. doi: 10.1128/jb.172.9.5140-5146.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klug G., Cohen S. N. Pleiotropic effects of localized Rhodobacter capsulatus puf operon deletions on production of light-absorbing pigment-protein complexes. J Bacteriol. 1988 Dec;170(12):5814–5821. doi: 10.1128/jb.170.12.5814-5821.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klug G., Drews G. Construction of a gene bank of Rhodopseudomonas capsulata using a broad host range DNA cloning system. Arch Microbiol. 1984 Nov;139(4):319–325. doi: 10.1007/BF00408373. [DOI] [PubMed] [Google Scholar]
  17. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lundberg U., von Gabain A., Melefors O. Cleavages in the 5' region of the ompA and bla mRNA control stability: studies with an E. coli mutant altering mRNA stability and a novel endoribonuclease. EMBO J. 1990 Sep;9(9):2731–2741. doi: 10.1002/j.1460-2075.1990.tb07460.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melefors O., von Gabain A. Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA. Cell. 1988 Mar 25;52(6):893–901. doi: 10.1016/0092-8674(88)90431-x. [DOI] [PubMed] [Google Scholar]
  21. Morgan W. D., Bear D. G., Litchman B. L., von Hippel P. H. RNA sequence and secondary structure requirements for rho-dependent transcription termination. Nucleic Acids Res. 1985 May 24;13(10):3739–3754. doi: 10.1093/nar/13.10.3739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morgan W. D., Bear D. G., von Hippel P. H. Rho-dependent termination of transcription. II. Kinetics of mRNA elongation during transcription from the bacteriophage lambda PR promoter. J Biol Chem. 1983 Aug 10;258(15):9565–9574. [PubMed] [Google Scholar]
  23. Newbury S. F., Smith N. H., Higgins C. F. Differential mRNA stability controls relative gene expression within a polycistronic operon. Cell. 1987 Dec 24;51(6):1131–1143. doi: 10.1016/0092-8674(87)90599-x. [DOI] [PubMed] [Google Scholar]
  24. Nilsson G., Belasco J. G., Cohen S. N., von Gabain A. Effect of premature termination of translation on mRNA stability depends on the site of ribosome release. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4890–4894. doi: 10.1073/pnas.84.14.4890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nilsson G., Lundberg U., von Gabain A. In vivo and in vitro identity of site specific cleavages in the 5' non-coding region of ompA and bla mRNA in Escherichia coli. EMBO J. 1988 Jul;7(7):2269–2275. doi: 10.1002/j.1460-2075.1988.tb03067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schmidhauser T. J., Helinski D. R. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Suissa M. Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983 Sep;133(2):511–514. doi: 10.1016/0003-2697(83)90117-3. [DOI] [PubMed] [Google Scholar]
  28. Youvan D. C., Bylina E. J., Alberti M., Begusch H., Hearst J. E. Nucleotide and deduced polypeptide sequences of the photosynthetic reaction-center, B870 antenna, and flanking polypeptides from R. capsulata. Cell. 1984 Jul;37(3):949–957. doi: 10.1016/0092-8674(84)90429-x. [DOI] [PubMed] [Google Scholar]
  29. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES