Summary
Escherichia coli was depleted of ribosomes by a thermal shock at 47° C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30° C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.
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Abbreviations
- SSC:
-
0.15 M Nacl, 0.015 M sodium citrate (pH 7.0)
- mRNA:
-
messenger ribonucleic acid
- IPTG:
-
isopropyl-β-D-thiogalactopyranoside
- cAMP:
-
adenosine 3′: 5′-cyclic monophosphoric acid
- LDS:
-
lithium dodecyl sulfate
- TCA:
-
trichloroacetic acid
References
Adesnik, M., Levinthal, C.: The synthesis and degradation of lactose operon messenger RNA in E. coli. Cold Spring Harbor Symp. Quant. Biol. 35, 451–459 (1970)
Adhya, S., Gottesman, M., Crombugghe, de B.: Release of polarity in Escherichia coli by gene N of phage λ: termination and antitermination of transcription. Proc. Natl. Acad. Sci. U.S.A. 71, 2543–2538 (1974)
Adhya, S., Gottesman, M., Crombugghe, de B., Court, D.: Transcription termination regulates gene expression. In: RNA polymerase (Losick, R., Chamberlin, M., eds.) pp. 719–730 New York: Cold Spring Harbor Laboratory 1976
Beckwith, J.: Restoration of operon activity by suppressors. Biochim. Biophys. Acta 76, 162–164 (1963)
Contesse, G., Crepin, M., Gros, F.: Transcription of the lactose operon in E. coli. In: The lactose operon (Beckwith, J.R., Zipser D., eds.), pp. 111–141. New York: Cold Spring Harbor Laboratory 1970
Crombugghe, de B., Adhya, S., Gottesman, M., Pastan, I.: Effect of rho on transcription of bacterial operons. Nature New Biol. 241 260–264 (1973)
Davis, B.D., Mingioli, E.S.: Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60, 17–28 (1950)
Franklin, N.C.: Altered reading of genetic signals fused to the N operon of bacteriophage λ: genetic evidence for modification of poymerase by the protein product of the N gene. J. Mol. Biol. 89 33–48 (1974)
Franklin, N.C., Luria, S.E.: Transduction by bacteriophage P1 and the properties of the lac genetic region in E. coli and S. dysenteriae. Virology 15, 299–311 (1961)
Franklin, N.C., Yanofsky, C.: The N protein of λ: Evidence bearing on transcription termination, polarity and the alteration of E. coli RNA polymerase. In: RNA polymerase (Losick, R., Chamberlin, M., eds.) pp. 693–706, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1976
Gillespie, D., Spiegelman, S.: A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829–842 (1965)
Gupta, R.S., Schlessinger, D.: Differential modes of chemical decay for early and late Lambda messenger RNA. J. Mol. Biol. 92, 311–318 (1975)
Har-El, R., Silberstein, A., Kuhn, J., Tal, M.: Synthesis and degradation of lac mRNA in E. coli depleted of 30S ribosomal subunits. Mol. Gen. Genet. 173, 135–144 (1979)
Imamoto, F., Kano, Y.: Inhibition of transcription of the tryptophan operon in Escherichia coli by a block in initiation of translation. Nature New Biol. 232, 169–173 (1971)
Imamoto, F., Yanofsky, C.: Transcription of the tryptophan operon in polarity mutants of Escherichia coli. I. Characterization of the tryptophan messenger RNA of polar mutants. J. Mol. Biol. 28, 1–23 (1967a)
Imamoto, F., Yanofsky, C.: Transcription of the tryptophan operon in polarity mutants of Escherichia coli. II. Evidence for normal production of trp-mRNA molecules and for permature termination of transcription. J. Mol. Biol. 28, 25–35 (1967b)
Jacob, F., Monod, J.: Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356 (1961)
Jacobs, K.A., Shen, V., Schlessinger, D.: Coupling of lac mRNA transcription to translation in Escherichia coli cell extracts. Proc. Natl. Acad. Sci. U.S.A. 75, 158–161 (1978)
Korn, L.J., Yanofsky, C.: Polarity suppressors defective in transcription termination at the attenuator of the tryptophan operon of Escherichia coli have altered Rho factor. J. Mol. Biol. 106, 231–241 (1976)
Kourilsky, P., Bourguignon, M.F., Gros, F.: Kinetics of viral transcription after induction of prophage. In: Bacteriophage Lambda (A.D. Hershey, ed.), pp. 647–666. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1971
Martin, R.G., Talal, N.: Translation and polarity in the histidine operon. IV. Relation of polarity to map position in his C. J. Mol. Biol. 36, 219–229 (1968)
Miller, J.H.: Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory 1972
Morrison, S.L., Zipser, D.: Polypeptide products of nonsense mutations I. Termination fragments from nonsense mutations in the Z gene of the lac operon of Escherichia coli. J. Mol. Biol. 50, 359–371 (1970)
Morse, D.E., Guertin, M.: Regulation of mRNA utilization and degradation by amino acid starvation. Nature New Biol. 232, 165–169 (1971)
Morse, D.E., Yanofsky, C.: Polarity and the degradation of mRNA. Nature 224, 329–331 (1969)
Newton, A.: Re-initiation of polypeptide synthesis and polarity in the lac operon of Escherichia coli. J. Mol. Biol. 41, 329–339 (1969)
Newton, A., Beckwith, J., Zipser, D., Brenner, S.: Nonsense mutants and polarity in the lac operon of Escherichia coli. J. Mol. Biol. 14, 290–295 (1965)
Revel, M., Herzberg, M., Greenshpan, H.: Initiator protein dependent binding of messenger RNA to the ribosome. Cold Spring Harbor Symp. Quant. Biol. 34, 261–275 (1969)
Richardson, J.P.: RNA synthesis termination factor Rho. FEBS 11th meeting Copenhagen, In: Gene expression (Clark, B.F.C., Klenow, H., Zeuthen, J., eds.), pp. 153–162. Oxford, Pergamon Press (1977)
Richardson, J.P., Grimley, C., Lowery, C.: Transcription termination factor Rho activity is altered in Escherichia coli with suA mutations. Proc. Natl. Acad. Sci. U.S.A. 72, 1725–1728 (1975)
Roberts, J.W.: Termination factor for RNA synthesis. Nature 224, 1168–1174 (1969)
Ron, E.Z., Davis, B.D.: Growth rate of Escherichia coli at elevated temperatures: Limitation by methionine. J. Bacteriol. 107, 391–396 (1971)
Sarabhai, A.S., Stutton, A.O.W., Brenner, S., Bolle, A.: Colinearity of the gene with the polypeptide chain. Nature 201, 13–17 (1964)
Stent, G.S.: Genetic transcription. Proc. R. Soc. Lond. [Biol.] 164, 181–197 (1966)
Tal, M., Silberstein, A., Myner, K.: In vivo reassembly of 30S ribosomal subunits following their specific destruction by thermal shock. Biochim. Biophys. Acta. 479, 479–496 (1977)
Varmus, H.E., Perlman, R.L., Pastan, I.: Regulation of lac transcription in antibiotic-treated E. coli. Nature New Biol. 230, 41–44 (1971)
Yanofsky, C., Ito, J.: Nonsense codons and polarity in the tryptophan operon. J. Mol. Biol. 21, 313–334 (1966)
Zipser, D., Zabell, S., Rothman, J., Grodzicker, T., Wenk, M.: Fine structure of the gradient of polarity in the Z gene of the lac operon of Escherichia coli. J. Mol. Biol. 49, 251–254 (1970)
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Communicated by H.G. Wittmann
The paper forms part of the first author's M.Sc. thesis
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Cohen, T., Silberstein, A., Kuhn, J. et al. Relief of polarity in E. coli depleted of 30S ribosomal subunits. Molec. gen. Genet. 173, 127–134 (1979). https://doi.org/10.1007/BF00330302
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DOI: https://doi.org/10.1007/BF00330302