Summary
Various synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the intact lac operon resulted in similar reductions of mRNA stability. The sequence alterations also reduced the translational efficiency and transcription through lacZ as monitored by measurements of galactokinase synthesis from the downstream galK gene. There was no correlation between the average translational frequency and the stability of the lacZ message indicating that some of the inserted sequences reduced mRNA stability directly and not as a consequence of their effect on translation. The reduction of transcription through the lacZ gene correlated with the reduction of translation in agreement with current models of transcriptional polarity.
Similar content being viewed by others
References
Atkins JF, Nichols BP, Thompson S (1983) The nucleotide sequence of the first externally suppressible — 1 frameshift mutant, and of some nearby leaky frameshift mutants. EMBO J 2:1345–1350
Belasco JG, Nilsson G, von Gabain A, Cohen SN (1986) The stability of E. coli gene transcripts is dependent on determinants localized to specific mRNA segments. Cell 46:245–251
Bolivar F, Rodriguez RL, Greene PY, Betlach MC, Heynecker HL, Boyer HW, Crosa YH, Falkow S (1977) Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2:95–113
Bonekamp F, Andersen HD, Christensen T, Jensen KF (1985) Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation. Nucleic Acids Res 13:4113–4123
Büchel DE, Gronenborn B, Müller-Hill B (1980) Sequence of the lactose permease gene. Nature 283:541–545
Cannistraro VJ, Kennell D (1979) Escherichia coli lac operator mRNA affects translation initiation of β-galactosidase mRNA. Nature 277:407–409
Cannistraro VJ, Kennell D (1985) Evidence that the 5′ end of lac mRNA starts to decay as soon as it is synthesized. J Bacteriol 161:820–822
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207
Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980
Chanda PK, Ono M, Kuwano M, Kung H-F (1985) Cloning, sequence analysis, and expression of alteration of the mRNA stability gene (ams +) of Escherichia coli. J Bacteriol 161:446–449
Clark B, Maaløe O (1967) DNA replication and the division cycle in Escherichia coli. J Mol Biol 23:99–112
Cole JR, Nomura M (1986) Changes in the half-life of ribosomal protein messenger RNA caused by translational repression. J Mol Biol 188:383–392
Dalbow DG, Young R (1975) Synthesis time of β-galactosidase in Escherichia coli B/r as a function of growth rate. Biochem J 150:13–20
Debouck C, Riccio A, Schumperli D, McKenney K, Jeffers J, Hughes C, Rosenberg M (1985) Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon. Nucleic Acids Res 13:1841–1853
Donovan WP, Kushner SR (1986) Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12. Proc Natl Acad Sci USA 83:120–124
Farabaugh PJ (1978) Sequence of the lacI gene. Nature 274:765–769
Fowler AV, Zabin I (1983) Purification, structure, and properties of hybrid β-galactosidase proteins. J Biol Chem 258:14354–14358
Gold L, Pribnow D, Schneider T, Shinedling S, Singer BW, Stormo G (1981) Translational initiation in prokaryotes. Annu Rev Microbiol 35:365–403
Hall MN, Gabay J, Débarbouillé M, Schwartz M (1982) A role for mRNA secondary structure in the control of translational initiation. Nature 295:616–618
Hediger MA, Johnson DF, Nierlich DP, Zabin I (1985) DNA sequence of the lactose operon: The lacA gene and the transcriptional termination region. Proc Natl Acad Sci USA 82:6414–6418
Jacobson AB, Good L, Simonetti J, Zuker M (1984) Some simple computational methods to improve the folding of large RNAs. Nucleic Acids Res 12:45–52
Jaquet M, Kepes A (1971) Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied by measurement of its β-galactosidase synthesizing capacity in vivo. J Mol Biol 60:453–472
Johnsen M (1984) JINN, an integrated software package for molecular geneticists. Nucleic Acids Res 12:657–664
Kalnins A, Otto K, Rüther U, Müller-Hill B (1983) Sequence of the lacZ gene of Escherichia coli. EMBO J 2:593–597
Kastelein RA, Remaut E, Fiers W, van Duin J (1982) Lysis gene expression of RNA phage MS2 depends on a frameshift during translation of the overlapping coat protein gene. Nature 295:35–41
Kennell D, Riezman H (1977) Transcription and translation initiation frequencies of the Escherichia coli lac operon. J Mol Biol 114:1–21
Kepes A (1963) Kinetics of induced enzyme synthesis. Determination of the mean life of galactosidase specific messenger DNA. Biochim Biophys Acta 76:293–309
Lim LW, Kennell D (1979) Models for decay of Escherichia coli lac messenger RNA and evidence for inactivating cleavages between its messages. J Mol Biol 135:369–390
Maizels N (1974) E. coli lactose operon ribosome binding site. Nature 249:647–649
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
McKenney K, Shimatake H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by E. coli RNA polymerase. In: Chirikjian JS, Papas TS (eds) Gene amplification and analysis, vol 2. Elsevier Science Publishing Inc, New York, pp 384–415
Mott JE, Galloway JL, Platt T (1985) Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3′ exonucleolytic processing after rho-dependent termination. EMBO J 4:1887–1891
Munson LM, Stormo GD, Niece RL, Reznikoff WS (1984) lacZ translation initiation mutations. J Mol Biol 177:663–683
Nierlich DP, Kwan C, Murakawa GG, Mahoney PA, Ung AW, Caprioglio D (1985) Intercistronic sites in the lac operon. In: Schaechter M, Neidhardt FC, Ingraham JL, Kjeldgaard NO (eds) The molecular biology of bacterial growth. Jones and Bartlett Publishers Inc, Boston, pp 185–113
Ono M, Kuwano M (1979) A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA. J Mol Biol 129:343–357
Pedersen S (1984) Escherichia coli ribosomes translate in vivo with variable rate. EMBO J 3:2895–2898
Pedersen S, Reeh S, Friesen JD (1978) Functional mRNA half lives in E. coli. Mol Gen Genet 166:329–336
Platt T, Bear DG (1983) Role of RNA polymerase pfactor, and ribosomes in transcription termination. In: Beckwith J, Davies J, Gallant J (eds) Gene function in prokaryotes. Cold Spring Harbor Laboratory Press, New York, pp 123–161
Puga A, Borrás M-T, Tessman ES, Tessman I (1973) Difference between functional and structural integrity of messenger RNA. Proc Natl Acad Sci USA 70:2171–2175
Queen CL, Korn LJ (1980) Computer analysis of nucleic acids and proteins. Methods Enzymol 65:595–609
Reznikoff WS (1984) Gene expression in microbes: The lactose operon model system. Symp Soc Gen Microbiol 36 II:195–218
Schmeissner U, McKenney K, Rosenberg M, Court D (1984) Removal of a terminator structure by RNA processing regulates int gene expression. J Mol Biol 176:39–53
Schneider E, Blundell M, Kennell D (1978) Translation and mRNA decay. Mol Gen Genet 160:121–129
Schwartz T, Craig E, Kennell D (1970) Inactivation and degradation of messenger ribonucleic acid from the lactose operon of Escherichia coli. J Mol Biol 54:299–311
Shultz J, Silhavy TJ, Berman ML, Fiil N, Emr SD (1982) A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery. Cell 31:227–235
Singer P, Nomura M (1985) Stability of ribosomal protein mRNA and translational feedback regulation in Escherichia coli. Mol Gen Genet 199:543–546
Stanssens P, Remaut E, Fiers W (1986) Inefficient translation initiation causes premature transcription termination in the lacZ gene. Cell 44:711–718
STueber D, Bujard H (1982) Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1:1399–1404
Sutcliffe JG (1979) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–99
Ullmann A, Jacob F, Monod J (1968) On the subunit structure of wild-type versus complemented β-galactosidase of Escherichia coli. J Mol Biol 32:1–13
Varenne S, Buc J, Lloubes R, Lazdunski C (1984) Translation is a non-uniform process. Effect of tRNA availability on the rate of elongation of nascent polypeptide chains. J Mol Biol 180:549–576
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
von Gabain A, Belasco JG, Schottel JL, Chang ACY, Cohen SN (1983) Decay of mRNA in Escherichia coli: Investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci USA 80:653–657
von Heijne G, Nilsson L, Blomberg C (1977) Translation and messenger RNA secondary structure. J Theor Biol 68:321–329
Wong HC, Chang S (1986) Identification of a positive retroregulator that stabilizes mRNAs in bacteria. Proc Natl Acad Sci USA 83:3233–3237
Yamamoto T, Suyama A, Mori N, Yokota T, Wada A (1985) Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control. FEBS Lett 181:377–380
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Petersen, C. The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site. Mol Gen Genet 209, 179–187 (1987). https://doi.org/10.1007/BF00329856
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00329856