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. 1978 Aug;135(2):528–541. doi: 10.1128/jb.135.2.528-541.1978

Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli.

V Talkad, D Achord, D Kennell
PMCID: PMC222413  PMID: 98520

Abstract

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.

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Selected References

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

  1. Achord D., Kennell D. Metabolism of messenger RNA from the gal operon of Escherichia coli. J Mol Biol. 1974 Dec 15;90(3):581–599. doi: 10.1016/0022-2836(74)90236-8. [DOI] [PubMed] [Google Scholar]
  2. Anderson E. H. Growth Requirements of Virus-Resistant Mutants of Escherichia Coli Strain "B". Proc Natl Acad Sci U S A. 1946 May;32(5):120–128. doi: 10.1073/pnas.32.5.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Apirion D., Neil J., Watson N. Consequences of losing ribonuclease III on the Escherichia coli cell. Mol Gen Genet. 1976 Mar 22;144(2):185–190. doi: 10.1007/BF02428107. [DOI] [PubMed] [Google Scholar]
  4. Apirion D., Neil J., Watson N. Revertants from RNase III negative strains of Escherichia coli. Mol Gen Genet. 1976 Dec 8;149(2):201–210. doi: 10.1007/BF00332890. [DOI] [PubMed] [Google Scholar]
  5. Apirion D., Watson N. Analysis of an Escherichia coli strain carrying physiologically compensating mutations one of which causes an altered ribonuclease 3. Mol Gen Genet. 1974;132(2):89–104. doi: 10.1007/BF00272175. [DOI] [PubMed] [Google Scholar]
  6. Apirion D., Watson N. Mapping and characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid. J Bacteriol. 1975 Oct;124(1):317–324. doi: 10.1128/jb.124.1.317-324.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Apirion D., Watson N. Ribonuclease III is involved in motility of Escherichia coli. J Bacteriol. 1978 Mar;133(3):1543–1545. doi: 10.1128/jb.133.3.1543-1545.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Apirion D., Watson N. Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA. Mol Gen Genet. 1975;136(4):317–326. doi: 10.1007/BF00341716. [DOI] [PubMed] [Google Scholar]
  9. Blundell M., Kennell D. Evidence for endonucleolytic attack in decay of lac messenger RNA in Escherichia coli. J Mol Biol. 1974 Feb 25;83(2):143–161. doi: 10.1016/0022-2836(74)90385-4. [DOI] [PubMed] [Google Scholar]
  10. Crouch R. J. Ribonuclease 3 does not degrade deoxyribonucleic acid-ribonucleic acid hybrids. J Biol Chem. 1974 Feb 25;249(4):1314–1316. [PubMed] [Google Scholar]
  11. Dickson R. C., Abelson J., Barnes W. M., Reznikoff W. S. Genetic regulation: the Lac control region. Science. 1975 Jan 10;187(4171):27–35. doi: 10.1126/science.1088926. [DOI] [PubMed] [Google Scholar]
  12. Dunn J. J., Studier F. W. Effect of RNAase III, cleavage on translation of bacteriophage T7 messenger RNAs. J Mol Biol. 1975 Dec 15;99(3):487–499. doi: 10.1016/s0022-2836(75)80140-9. [DOI] [PubMed] [Google Scholar]
  13. Dunn J. J., Studier F. W. Processing transcription, and translation of bacteriophage T7 messenger RNAs. Brookhaven Symp Biol. 1975 Jul;(26):267–276. [PubMed] [Google Scholar]
  14. Dunn J. J., Studier F. W. T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease 3. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3296–3300. doi: 10.1073/pnas.70.12.3296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dunn J. J., Studier F. W. T7 early RNAs are generated by site-specific cleavages. Proc Natl Acad Sci U S A. 1973 May;70(5):1559–1563. doi: 10.1073/pnas.70.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gegenheimer P., Watson N., Apirion D. Multiple pathways for primary processing of ribosomal RNA in Escherichia coli. J Biol Chem. 1977 May 10;252(9):3064–3073. [PubMed] [Google Scholar]
  17. Gilbert W., Maxam A. The nucleotide sequence of the lac operator. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3581–3584. doi: 10.1073/pnas.70.12.3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ginsburg D., Steitz J. A. The 30 S ribosomal precursor RNA from Escherichia coli. A primary transcript containing 23 S, 16 S, and 5 S sequences. J Biol Chem. 1975 Jul 25;250(14):5647–5654. [PubMed] [Google Scholar]
  19. Hercules K., Schweiger M., Sauerbier W. Cleavage by RNase 3 converts T3 and T7 early precursor RNA into translatable message. Proc Natl Acad Sci U S A. 1974 Mar;71(3):840–844. doi: 10.1073/pnas.71.3.840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Imamoto F. Evidence for premature termination of transcription of the tryptophan operon in polarity mutants of Escherichia coli. Nature. 1970 Oct 17;228(5268):232–235. doi: 10.1038/228232a0. [DOI] [PubMed] [Google Scholar]
  21. Jacquet M., Kepes A. Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied studied by measurement of its beta-galactosidase synthesizing capacity in vivo. J Mol Biol. 1971 Sep 28;60(3):453–472. doi: 10.1016/0022-2836(71)90181-1. [DOI] [PubMed] [Google Scholar]
  22. KEPES A. KINETICS OF INDUCED ENZYME SYNTHESIS. DETERMINATION OF THE MEAN LIFE OF GALACTOSIDASE-SPECIFIC MESSENGER RNA. Biochim Biophys Acta. 1963 Oct 15;76:293–309. [PubMed] [Google Scholar]
  23. 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]
  24. Kennell D., Talkad V. Messenger RNA potential and the delay before exponential decay of messages. J Mol Biol. 1976 Jun 14;104(1):285–298. doi: 10.1016/0022-2836(76)90014-0. [DOI] [PubMed] [Google Scholar]
  25. Kindler P., Keil T. U., Hofschneider P. H. Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli. Mol Gen Genet. 1973 Oct 16;126(1):53–59. doi: 10.1007/BF00333481. [DOI] [PubMed] [Google Scholar]
  26. Kramer R. A., Rosenberg M., Steitz J. A. Nucleotide sequences of the 5' and 3' termini of bacteriophage T7 early messenger RNAs synthesized in vivo: evidence for sequence specificity in RNA processing. J Mol Biol. 1974 Nov 15;89(4):767–776. doi: 10.1016/0022-2836(74)90051-5. [DOI] [PubMed] [Google Scholar]
  27. Leive L., Kollin V. Synthesis, utilization and degradation of lactose operon mRNA in Escherichia coli. J Mol Biol. 1967 Mar 14;24(2):247–259. doi: 10.1016/0022-2836(67)90330-0. [DOI] [PubMed] [Google Scholar]
  28. Lozeron H. A., Anevski P. J., Apirion D. Antitermination and absence of processing of the leftward transcript of coliphage lambda in the RNAase III-deficient host. J Mol Biol. 1977 Jan 15;109(2):359–365. doi: 10.1016/s0022-2836(77)80039-9. [DOI] [PubMed] [Google Scholar]
  29. Maizels N. M. The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3585–3589. doi: 10.1073/pnas.70.12.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morse D. E., Mosteller R. D., Yanofsky C. Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spring Harb Symp Quant Biol. 1969;34:725–740. doi: 10.1101/sqb.1969.034.01.082. [DOI] [PubMed] [Google Scholar]
  31. Nikolaev N., Silengo L., Schlessinger D. Synthesis of a large precursor to ribosomal RNA in a mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3361–3365. doi: 10.1073/pnas.70.12.3361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pastushok C., Kennell D. Residual polarity and transcription-translation coupling during recovery from chloramphenicol or fusidic acid. J Bacteriol. 1974 Feb;117(2):631–640. doi: 10.1128/jb.117.2.631-640.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Revel M., Herzberg M., Greenshpan H. Initiator protein dependent binding of messenger RNA to the ribosome. Cold Spring Harb Symp Quant Biol. 1969;34:261–275. doi: 10.1101/sqb.1969.034.01.032. [DOI] [PubMed] [Google Scholar]
  34. Richardson J. P., Grimley C., Lowery C. Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations. Proc Natl Acad Sci U S A. 1975 May;72(5):1725–1728. doi: 10.1073/pnas.72.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Robertson H. D., Dickson E., Dunn J. J. A nucleotide sequence from a ribonuclease III processing site in bacteriophage T7 RNA. Proc Natl Acad Sci U S A. 1977 Mar;74(3):822–826. doi: 10.1073/pnas.74.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robertson H. D., Dunn J. J. Ribonucleic acid processing activity of Escherichia coli ribonuclease III. J Biol Chem. 1975 Apr 25;250(8):3050–3056. [PubMed] [Google Scholar]
  37. Robertson H. D., Webster R. E., Zinder N. D. Purification and properties of ribonuclease III from Escherichia coli. J Biol Chem. 1968 Jan 10;243(1):82–91. [PubMed] [Google Scholar]
  38. Rosenberg M., Kramer R. A. Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA. Proc Natl Acad Sci U S A. 1977 Mar;74(3):984–988. doi: 10.1073/pnas.74.3.984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rosenberg M., Kramer R. A., Steitz J. A. T7 early messenger RNAs are the direct products of ribonuclease III cleavage. J Mol Biol. 1974 Nov 15;89(4):777–782. doi: 10.1016/0022-2836(74)90052-7. [DOI] [PubMed] [Google Scholar]
  40. Schwartz T., Craig E., Kennell D. Inactivation and degradation of messenger ribnucleic acid from the lactose operon of Escherichia coli. J Mol Biol. 1970 Dec 14;54(2):299–311. doi: 10.1016/0022-2836(70)90431-6. [DOI] [PubMed] [Google Scholar]
  41. Schweitz H., Ebel J. P. A study of the mechanism of action of E. coli ribonuclease 3. Biochimie. 1971;53(5):585–593. doi: 10.1016/s0300-9084(71)80014-7. [DOI] [PubMed] [Google Scholar]
  42. Silengo L., Nikolaev N., Schlessinger D., Imamoto F. Stabilization of mRNA with polar effects in an Escherichia coli mutant. Mol Gen Genet. 1974;134(1):7–19. doi: 10.1007/BF00332808. [DOI] [PubMed] [Google Scholar]
  43. Smith T. F., Sadler J. R. The nature of lactose operator constitive mutations. J Mol Biol. 1971 Jul 28;59(2):273–305. doi: 10.1016/0022-2836(71)90051-9. [DOI] [PubMed] [Google Scholar]
  44. Sprague K. U., Steitz J. A. The 3' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions. Nucleic Acids Res. 1975 Jun;2(6):787–798. doi: 10.1093/nar/2.6.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Studier F. W. Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli. J Bacteriol. 1975 Oct;124(1):307–316. doi: 10.1128/jb.124.1.307-316.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

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