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. 1978 Mar;133(3):1351–1357. doi: 10.1128/jb.133.3.1351-1357.1978

In vitro synthesis of cystathionine gamma-synthetase in Escherichia coli K-12.

J H Krueger, J R Johnson, R C Greene
PMCID: PMC222172  PMID: 346568

Abstract

Synthesis of cystathionine gamma-synthetase directed by DNA from a lambdadmet transducing phage has been achieved in cell extracts from Escherichia coli K-12. Enzyme synthesis was stimulated two- to threefold by the addition of guanosine 3'-diphosphate 5'-diphosphate to the incubation mixtures. Kinetic studies showed a 1.5- to 2.0-min lag between initiation of transcription and completion of a translatable message. This lag is shorter than that observed for beta-galactosidase synthesis with DNA from a lac transducing phage known to initiate transcription at the lac promoter. This result, together with information on the structure of the transducing phage, shows that pL is not used for initiation of in vitro metB transcription. Attempts to demonstrate repression were not successful, and unexpectedly, extracts from metJ+ strains were found to be more effective at enzyme synthesis than those from their metJ derivatives.

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Selected References

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

  1. Bachmann B. J., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. COOK A., LEDERBERG J. Recombination studies of lactose nonfermenting mutants of Escherichia coli K-12. Genetics. 1962 Oct;47:1335–1353. doi: 10.1093/genetics/47.10.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cashel M. Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes. Anal Biochem. 1974 Jan;57(1):100–107. doi: 10.1016/0003-2697(74)90056-6. [DOI] [PubMed] [Google Scholar]
  4. De Crombrugghe B., Chen B., Gottesman M., Pastan I., Varmus H. E., Emmer M., Perlman R. L. Regulation of lac mRNA synthesis in a soluble cell-free system. Nat New Biol. 1971 Mar 10;230(10):37–40. doi: 10.1038/newbio230037a0. [DOI] [PubMed] [Google Scholar]
  5. Greene R. C., Hunter J. S., Coch E. H. Properties of metK mutants of Escherichia coli K-12. J Bacteriol. 1973 Jul;115(1):57–67. doi: 10.1128/jb.115.1.57-67.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Greene R. C. Improved procedure for synthesis of radioactive O-succinyl-L-homoserine. Anal Biochem. 1977 Mar;78(1):182–187. doi: 10.1016/0003-2697(77)90022-7. [DOI] [PubMed] [Google Scholar]
  7. Greene R. C., Su C. H., Holloway C. T. S-Adenosylmethionine synthetase deficient mutants of Escherichia coli K-12 with impaired control of methionine biosynthesis. Biochem Biophys Res Commun. 1970 Mar 27;38(6):1120–1126. doi: 10.1016/0006-291x(70)90355-4. [DOI] [PubMed] [Google Scholar]
  8. Guterman S. K., Dann L. Excretion of enterochelin by exbA and exbB mutants of Escherichia coli. J Bacteriol. 1973 Jun;114(3):1225–1230. doi: 10.1128/jb.114.3.1225-1230.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hobson A. C., Smith D. A. S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium. Mol Gen Genet. 1973 Oct 16;126(1):7–18. doi: 10.1007/BF00333477. [DOI] [PubMed] [Google Scholar]
  10. Holloway C. T., Greene R. C., Su C. H. Regulation of S-adenosylmethionine synthetase in Escherichia coli. J Bacteriol. 1970 Nov;104(2):734–747. doi: 10.1128/jb.104.2.734-747.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hunter J. S., Greene R. C., Su C. H. Genetic characterization of the metK locus in Escherichia coli K-12. J Bacteriol. 1975 Jun;122(3):1144–1152. doi: 10.1128/jb.122.3.1144-1152.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs K. A., Schlessinger D. Escherichia coli DNA-directed beta-galactosidase synthesis in presence and absence of Ca2+. Biochemistry. 1977 Mar 8;16(5):914–920. doi: 10.1021/bi00624a016. [DOI] [PubMed] [Google Scholar]
  13. Johnson J. R., Greene R. C., Krueger J. H. Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster. J Bacteriol. 1977 Sep;131(3):795–800. doi: 10.1128/jb.131.3.795-800.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. KATZEN H. M., BUCHANAN J. M. ENZYMATIC SYNTHESIS OF THE METHYL GROUP OF METHIONINE. 8. REPRESSION-DEREPRESSION, PURIFICATION, AND PROPERTIES OF 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE FROM ESCHERICHIA COLI. J Biol Chem. 1965 Feb;240:825–835. [PubMed] [Google Scholar]
  15. Kung H. F., Spears C., Greene R. C., Weissbach H. Regulation of the terminal reactions in methionine biosynthesis by vitamin B 12 and methionine. Arch Biochem Biophys. 1972 May;150(1):23–31. doi: 10.1016/0003-9861(72)90005-7. [DOI] [PubMed] [Google Scholar]
  16. Lawrence D. A., Smith D. A., Rowbury R. J. Regulation of methionine synthesis in Salmonella typhimurium: mutants resistant to inhibition by analogues of methionine. Genetics. 1968 Apr;58(4):473–492. doi: 10.1093/genetics/58.4.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lederman M., Zubay G. DNA directed peptide synthesis. V. The cell-free synthesis of a polypeptide with beta-galactosidase activity. Biochem Biophys Res Commun. 1968 Aug 21;32(4):710–714. doi: 10.1016/0006-291x(68)90297-0. [DOI] [PubMed] [Google Scholar]
  18. Milner L., Whitfield C., Weissbach H. Effect of L-methionine and vitamin B 12 on methionine biosynthesis in Escherichia coli. Arch Biochem Biophys. 1969 Sep;133(2):413–419. doi: 10.1016/0003-9861(69)90470-6. [DOI] [PubMed] [Google Scholar]
  19. PATTERSON M. S., GREENE R. C. MEASUREMENT OF LOW ENERGY BETA-EMITTERS IN AQUEOUS SOLUTION BY LIQUID SCINTILLATION COUNTING OF EMULSIONS. Anal Chem. 1965 Jun;37:854–857. doi: 10.1021/ac60226a017. [DOI] [PubMed] [Google Scholar]
  20. Patte J. C., Le Bras G., Cohen G. N. Regulation by methionine of the synthesis of a third aspartokinase and of a second homoserine dehydrogenase in Escherichia coli K 12. Biochim Biophys Acta. 1967 Mar 22;136(2):245–247. doi: 10.1016/0304-4165(67)90069-4. [DOI] [PubMed] [Google Scholar]
  21. Rowbury R. J., Woods D. D. The regulation of cystathionine formation in Escherichia coli. J Gen Microbiol. 1966 Jan;42(1):155–163. doi: 10.1099/00221287-42-1-155. [DOI] [PubMed] [Google Scholar]
  22. SCHLENK F., DAINKO J. L., STANFORD S. M. Improved procedure for the isolation of S-adenosylmethionine and S-adenosylethionine. Arch Biochem Biophys. 1959 Jul;83(1):28–34. doi: 10.1016/0003-9861(59)90006-2. [DOI] [PubMed] [Google Scholar]
  23. Savin M. A., Flavin M., Slaughter C. Regulation of homocysteine biosynthesis in Salmonella typhimurium. J Bacteriol. 1972 Aug;111(2):547–556. doi: 10.1128/jb.111.2.547-556.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shapiro S. K., Ehninger D. J. Methods for the analysis and preparation of adenosylmethionine and adenosylhomocysteine. Anal Biochem. 1966 May;15(2):323–333. doi: 10.1016/0003-2697(66)90038-8. [DOI] [PubMed] [Google Scholar]
  25. Smolin D. E., Umbarger H. E. Specificity of the stimulation of in vitro ribonucleic acid synthesis by guanosine 5'-diphosphate 3'-diphosphate. Mol Gen Genet. 1975 Dec 9;141(4):277–284. doi: 10.1007/BF00331449. [DOI] [PubMed] [Google Scholar]
  26. Somerville C. R., Ahmed A. rel-dependent methionine requirement in revertants of a methionyl-transfer RNA synthetase mutant of Escherichia coli. J Mol Biol. 1977 Mar 25;111(1):77–81. doi: 10.1016/s0022-2836(77)80133-2. [DOI] [PubMed] [Google Scholar]
  27. Stephens J. C., Artz S. W., Ames B. N. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino-acid deficiency. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4389–4393. doi: 10.1073/pnas.72.11.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Su C. H., Greene R. C. Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ plus-metJ minus diploid. Proc Natl Acad Sci U S A. 1971 Feb;68(2):367–371. doi: 10.1073/pnas.68.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zubay G., Chambers D. A. A DNA-directed cell-free system for beta-galactosidase synthesis; characterization of the de novo synthesized enzyme and some aspects of the regulation of synthesis. Cold Spring Harb Symp Quant Biol. 1969;34:753–761. doi: 10.1101/sqb.1969.034.01.085. [DOI] [PubMed] [Google Scholar]
  30. Zubay G. In vitro synthesis of protein in microbial systems. Annu Rev Genet. 1973;7:267–287. doi: 10.1146/annurev.ge.07.120173.001411. [DOI] [PubMed] [Google Scholar]

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