1887

Abstract

The mutation of the double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in . Here we show that is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in . Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6·85 for RNAaseIII-G97E and 7·3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg limitation. The hypothesis that limitation of Mg ions is responsible for the phenotypes of the mutation in and is discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-4-717
1993-04-01
2021-05-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/4/mic-139-4-717.html?itemId=/content/journal/micro/10.1099/00221287-139-4-717&mimeType=html&fmt=ahah

References

  1. Altuvia S., Kornitzer D., Kobi S., Oppenheim A. B. 1991; Functional and structural elements of the mRNA of the cIII gene of bacteriophage lambda. Journal of Molecular Biology 218:723–733
    [Google Scholar]
  2. Apirion D., Watson N. 1975; Mapping and characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid. Journal of Bacteriology 124:317–324
    [Google Scholar]
  3. Bardwell J. C. A., Regnier P., Chen S., Nakamura Y., Grunberg-Manago M., Court D. L. 1989; Autoregulation of RNaseIII operon by mRNA processing. EMBO Journal 8:3401–3407
    [Google Scholar]
  4. Davidov Y., Zivion G., Pines O. 1992; Ribonuclease III reduces the efficiency of bacteriophage gyl propagation in E. coli. Current Microbiology 24:63–66
    [Google Scholar]
  5. Dunn J. J. 1976; RNaseIII cleavage of single-stranded RNA. Journal of Biological Chemistry 251:3807–3814
    [Google Scholar]
  6. Dunn J. J., Studier F. W. 1973; T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease III. Proceedings of the National Academy of Sciences of the United States of America 70:3296–3300
    [Google Scholar]
  7. Dunn J. J., Studier F. W. 1975; Effect of RNaseIII cleavage on translation of bacteriophage T7 messenger RNAs. Journal of Molecular Biology 99:487–499
    [Google Scholar]
  8. Kindler P., Keil T. U., Hoffschneider P. H. 1973; Isolation and characterization of a ribonuclease III deficient mutant of Escherichia coli. Molecular and General Genetics 126:53–69
    [Google Scholar]
  9. King T. C., Sirdeshmukh R., Schlessinger D. 1986; Nucleolytic processing of ribonucleic acid transcripts in prokaryotes. Microbiological Reviews 50:428–451
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  11. Maguire M. E. 1990; Magnesium, a regulated and regulatory cation. In Metal Ions in Biological Systems 26 pp. 135–153 Sigel H., Sigel A. Edited by New York: Dekker;
    [Google Scholar]
  12. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  13. March P. E., Gonzalez M. A. 1990; Characterization of the biochemical properties of recombinant ribonuclease III. Nucleic Acids Research 18:3293–3298
    [Google Scholar]
  14. March P. E., Ahnn T., Inouye M. 1985; The DNA sequence of the gene rnc encoding ribonuclease III of Escherichia coli. Nucleic Acids Research 13:4677–4685
    [Google Scholar]
  15. Masui Y., Coleman J., Inouye M. 1983; Multipurpose expression cloning vehicles in E. coli. In Experimental Manipulation of Gene Expression pp. 15–32 Inouye M. Edited by Epidemiological and Molecular Aspects on Cholera New York: Academic Press;
    [Google Scholar]
  16. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  17. Nashimoto H., Uchida H. 1985; DNA sequencing of the E. coli ribonuclease III gene and its mutations. Molecular and General Genetics 201:25–29
    [Google Scholar]
  18. Pines O., Yoon H. J., Inouye M. 1988; Expression of doublestranded RNA-specific RNaseIII of Escherichia coli is lethal to Saccharomyces cerevisiae. Journal of Bacteriology 170:2989–2993
    [Google Scholar]
  19. Portier C., Dondon L., Grunberg-Manago M., Regnier P. 1987; The first step in the functional activation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5´ end. EMBO Journal 6:2165–2170
    [Google Scholar]
  20. Robertson H. D., Dunn J. J. 1975; Ribonucleic acid processing activity of Escherichia coli ribonuclease III cleavage sites. Journal of Biological Chemistry 250:3050–3056
    [Google Scholar]
  21. Robertson H. D., Webster R. E., Zinder N. D. 1968; Purification and properties of ribonuclease III from Escherichia coli. Journal of Biological Chemistry 243:82–91
    [Google Scholar]
  22. Rosenberg A. H., Lade B. N., Chui D., Lin S., Dunn J. J., Studier F. W. 1987; Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125–135
    [Google Scholar]
  23. Shonberger O., Hirst T. R., Pines O. 1991; Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae. Molecular Microbiology 5:2663–2671
    [Google Scholar]
  24. Sillero A., Ribeiro J. H. 1989; Isolectric points of proteins: theoretical determination. Analytical Biochemistry 179:319–325
    [Google Scholar]
  25. Snavely M. D. 1990; Magnesium transport in prokaryotic cells.. In Metal Ions in Biological Systems 26 pp. 155–175 Sigel H., Sigel A. Edited by New York: Dekker;
    [Google Scholar]
  26. Studier F. W. 1975; Genetic mapping of a mutation that causes ribonuclease III deficiency in Escherichia coli. Journal of Bacteriology 124:307–316
    [Google Scholar]
  27. Studier F. W., Dunn J. J. 1983; Complete nucleotide sequence of bacteriophage T7 DNA and the location of T7 genetic elements. Journal of Molecular Biology 166:477–535
    [Google Scholar]
  28. Tariff H. E., Chen S. M., Court D. L. 1989; Genetic analysis of the me operon of Escherichia coli. Journal of Bacteriology 171:2581–2590
    [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76:4350–4354
    [Google Scholar]
  30. Watson N., Apirion D. 1985; Molecular cloning of the gene for the RNA-processing enzyme RNaseIII of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 82:849–853
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-4-717
Loading
/content/journal/micro/10.1099/00221287-139-4-717
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error