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. 1993 Apr;175(8):2327–2333. doi: 10.1128/jb.175.8.2327-2333.1993

Mutational analysis of segmental stabilization of transcripts from the Zymomonas mobilis gap-pgk operon.

G Burchhardt 1, K F Keshav 1, L Yomano 1, L O Ingram 1
PMCID: PMC204521  PMID: 8468293

Abstract

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are transcribed together from the gap-pgk operon. However, higher levels of the former enzyme are present in the cytoplasm because of increased stability of a 5' segment containing the gap coding region. This segment is bounded by an upstream untranslated region which can be folded into many stem-loop structures and a prominent intercistronic stem-loop. Mutations eliminating a proposed stem-loop in the untranslated region or the intercistronic stem-loop resulted in a decrease in the stability and pool size of the 5' gap segment. Site-specific mutations in the unpaired regions of both of these stems also altered the message pools. Elimination of the intercistronic stem appeared to reduce the endonucleolytic cleavage within the pgk coding region, increasing the stability and abundance of the full-length message. DNA encoding the prominent stem-loop at the 3' end of the message was shown to be a transcriptional terminator both in Z. mobilis and in Escherichia coli. This third stem-loop region (part of the transcriptional terminator) was required to stabilize the full-length gap-pgk message.

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Selected References

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  1. Ahmad I., Finkelstein J. A., Steggles A. W. The analysis of RNA by in situ agarose gel hybridization is more sensitive than the equivalent northern blot analysis. Biotechniques. 1990 Feb;8(2):162–165. [PubMed] [Google Scholar]
  2. An H., Scopes R. K., Rodriguez M., Keshav K. F., Ingram L. O. Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein. J Bacteriol. 1991 Oct;173(19):5975–5982. doi: 10.1128/jb.173.19.5975-5982.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnell W. O., Liu J., Hesman T. L., O'Neill M. C., Conway T. The Zymomonas mobilis glf, zwf, edd, and glk genes form an operon: localization of the promoter and identification of a conserved sequence in the regulatory region. J Bacteriol. 1992 May;174(9):2816–2823. doi: 10.1128/jb.174.9.2816-2823.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bechhofer D. H., Dubnau D. Induced mRNA stability in Bacillus subtilis. Proc Natl Acad Sci U S A. 1987 Jan;84(2):498–502. doi: 10.1073/pnas.84.2.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brawerman G. Determinants of messenger RNA stability. Cell. 1987 Jan 16;48(1):5–6. doi: 10.1016/0092-8674(87)90346-1. [DOI] [PubMed] [Google Scholar]
  6. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [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. Cannistraro V. J., Kennell D. RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli. J Bacteriol. 1991 Aug;173(15):4653–4659. doi: 10.1128/jb.173.15.4653-4659.1991. [DOI] [PMC free article] [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. Chen L. H., Emory S. A., Bricker A. L., Bouvet P., Belasco J. G. Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA. J Bacteriol. 1991 Aug;173(15):4578–4586. doi: 10.1128/jb.173.15.4578-4586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Conway T., Byun M. O., Ingram L. O. Expression Vector for Zymomonas mobilis. Appl Environ Microbiol. 1987 Feb;53(2):235–241. doi: 10.1128/aem.53.2.235-241.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conway T., Fliege R., Jones-Kilpatrick D., Liu J., Barnell W. O., Egan S. E. Cloning, characterization and expression of the Zymononas mobilis eda gene that encodes 2-keto-3-deoxy-6-phosphogluconate aldolase of the Entner-Doudoroff pathway. Mol Microbiol. 1991 Dec;5(12):2901–2911. doi: 10.1111/j.1365-2958.1991.tb01850.x. [DOI] [PubMed] [Google Scholar]
  13. Conway T., Osman Y. A., Ingram L. O. Gene expression in Zymomonas mobilis: promoter structure and identification of membrane anchor sequences forming functional lacZ' fusion proteins. J Bacteriol. 1987 Jun;169(6):2327–2335. doi: 10.1128/jb.169.6.2327-2335.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Conway T., Sewell G. W., Ingram L. O. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol. 1987 Dec;169(12):5653–5662. doi: 10.1128/jb.169.12.5653-5662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. DeHoff B. S., Lee J. K., Donohue T. J., Gumport R. I., Kaplan S. In vivo analysis of puf operon expression in Rhodobacter sphaeroides after deletion of a putative intercistronic transcription terminator. J Bacteriol. 1988 Oct;170(10):4681–4692. doi: 10.1128/jb.170.10.4681-4692.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deutscher M. P. Ribonucleases, tRNA nucleotidyltransferase, and the 3' processing of tRNA. Prog Nucleic Acid Res Mol Biol. 1990;39:209–240. doi: 10.1016/s0079-6603(08)60628-5. [DOI] [PubMed] [Google Scholar]
  17. Eddy C. K., Keshav K. F., An H., Utt E. A., Mejia J. P., Ingram L. O. Segmental message stabilization as a mechanism for differential expression from the Zymomonas mobilis gap operon. J Bacteriol. 1991 Jan;173(1):245–254. doi: 10.1128/jb.173.1.245-254.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eddy C. K., Mejia J. P., Conway T., Ingram L. O. Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis. J Bacteriol. 1989 Dec;171(12):6549–6554. doi: 10.1128/jb.171.12.6549-6554.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ehretsmann C. P., Carpousis A. J., Krisch H. M. mRNA degradation in procaryotes. FASEB J. 1992 Oct;6(13):3186–3192. doi: 10.1096/fasebj.6.13.1397840. [DOI] [PubMed] [Google Scholar]
  20. Emory S. A., Bouvet P., Belasco J. G. A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli. Genes Dev. 1992 Jan;6(1):135–148. doi: 10.1101/gad.6.1.135. [DOI] [PubMed] [Google Scholar]
  21. Gorski K., Roch J. M., Prentki P., Krisch H. M. The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts. Cell. 1985 Dec;43(2 Pt 1):461–469. doi: 10.1016/0092-8674(85)90176-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Koraimann G., Högenauer G. A stable core region of the tra operon mRNA of plasmid R1-19. Nucleic Acids Res. 1989 Feb 25;17(4):1283–1298. doi: 10.1093/nar/17.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mackenzie K. F., Conway T., Aldrich H. C., Ingram L. O. Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis. J Bacteriol. 1989 Sep;171(9):4577–4582. doi: 10.1128/jb.171.9.4577-4582.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mayford M., Weisblum B. Conformational alterations in the ermC transcript in vivo during induction. EMBO J. 1989 Dec 20;8(13):4307–4314. doi: 10.1002/j.1460-2075.1989.tb08617.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McLaren R. S., Newbury S. F., Dance G. S., Causton H. C., Higgins C. F. mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo. J Mol Biol. 1991 Sep 5;221(1):81–95. [PubMed] [Google Scholar]
  28. Mejia J. P., Burnett M. E., An H., Barnell W. O., Keshav K. F., Conway T., Ingram L. O. Coordination of expression of Zymomonas mobilis glycolytic and fermentative enzymes: a simple hypothesis based on mRNA stability. J Bacteriol. 1992 Oct;174(20):6438–6443. doi: 10.1128/jb.174.20.6438-6443.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Osman Y. A., Conway T., Bonetti S. J., Ingram L. O. Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation. J Bacteriol. 1987 Aug;169(8):3726–3736. doi: 10.1128/jb.169.8.3726-3736.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Owolabi J. B., Rosen B. P. Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon. J Bacteriol. 1990 May;172(5):2367–2371. doi: 10.1128/jb.172.5.2367-2371.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sandler P., Weisblum B. Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis. J Mol Biol. 1988 Oct 20;203(4):905–915. doi: 10.1016/0022-2836(88)90116-7. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Yamamoto T., Imamoto F. Differential stability of trp messenger RNA synthesized originating at the trp promoter and pL promoter of lambda trp phage. J Mol Biol. 1975 Feb 25;92(2):289–304. doi: 10.1016/0022-2836(75)90228-4. [DOI] [PubMed] [Google Scholar]
  35. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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