Summary
An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of β-galactosidase production. In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously. This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied. Increasing the trpR + gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator “sinks” result in significantly decreased levels of Trp repressor protein. Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements. The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system. Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess. The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels.
Similar content being viewed by others
Abbreviations
- bp:
-
base pairs
- XG:
-
5-bromo-4-chloro-3-indolyl-β-D-galactosidase
References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Blattner FR, Williams BG, Blechl AE, Denniston-Thompson HE, Faber HE, Furlong L-A, Grunwald DJ, Kiefer DO, Moore DD, Schumm JW, Sheldon EL, Smithies O (1977) Charon phages; safer derivatives of bacteriophage lambda for DNA cloning. Science 196:161–169
Bogosian G, Bertrand K, Somerville R (1981) Trp repressor protein controls its own structural gene. J Mol Biol 149:821–825
Bogosian G, Somerville RL (1983) Trp repressor protein is capable of intruding into other amino acid biosynthetic systems. Mol Gen Genet 191:51–58
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HW, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Brown KD (1968) Regulation of aromatic amino acid biosynthesis in Escherichia coli K-12. Genetics 60:31–48
Camakaris H, Pittard J (1974) Purification and properties of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) from Escherichia coli. J Bacteriol 120:590–597
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Close TJ, Rodriguez RL (1982) Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
Cohen G, Jacob F (1959) Sur la repression de la synthese des enzymes intervenant dans la formation du tryptophane chez Escherichia coli. Compt Rend Acad Sci Paris 248:3490–3492
Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Covarrubias L, Cervantes L, Covarrubias A, Soberon X, Vichido I, Blanco A, Kupersztoch-Portnoy YM, Bolivar F (1981) Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene 13:25–35
Epstein W (1967) Transposition of the lac region of Escherichia coli. IV. Escape from repression in bacteriophage-carried lac genes. J Mol Biol 30:529–543
Gottesman ME, Yarmolinsky MB (1968) Integration-negative mutants of bacteriophage lambda. J Mol Biol 31:487–505
Gunsalus RP, Yanofsky C (1980) Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc Natl Acad Sci USA 77:7117–7121
Gunsalus RP, Zurawski G, Yanofsky C (1979) Structural and functional analysis of cloned deoxyribonucleic acid containing the trpR-thr region of the Escherichia coli chromosome. J Bacteriol 140:106–113
Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl Acids Res 9:2989–2998
Kelley RL, Yanofsky C (1982) trp aporepressor production is controlled by autogenous regulation and inefficient translation. Proc Natl Acad Sci USA 79:3120–3124
Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Manson MD, Yanofsky C (1976) Tryptophan operon regulation in interspecific hybrids of enteric bacteria. J Bacteriol 126:679–689
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
McGeoch D, McGeoch J, Morse DE (1973) Synthesis of tryptophan operon RNA in a cell-free system. Nature New Biol 245:137–140
McKenney K, Shimatake H, Court D, Schmeissmer U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. Gene Amplif Anal 2:383–415
Messing J (1982) An integrative strategy of DNA sequencing and experiments beyond. Genetic engineering, principles and methods 4:19–35
Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucl Acids Res 9:309–321
Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Mitchell DH, Reznikoff WS, Beckwith JR (1975) Genetic fusions defining trp and lac operon regulatory elements. J Mol Biol 93:331–350
Morse DE, Yanofsky C (1969) Amber mutants of the trpR regulatory gene. J Mol Biol 44:185–193
Oppenheim DS, Bennett GN, Yanofsky C (1980) Escherichia coli RNA polymerase and trp repressor interaction with the promoter-operator region of the tryptophan operon of Salmonella typhimurium. J Mol Biol 144:133–142
Oxender DL, Zurawski G, Yanofsky C (1979) Attenuation in the Eschericia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region. Proc Natl Acad Sci USA 76:524–528
Roeder W, Somerville RL (1979) Cloning the trpR gene. Mol Gen Genet 176:361–368
Rose JK, Squires CL, Yanofsky C, Yang H-L, Zubay G (1973) Regulation of in vitro transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nature New Biol 245:133–137
Rose JK, Yanofsky C (1974) Interaction of the operator of the tryptophan operon with repressor. Proc Natl Acad Sci USA 71:3134–3138
Russell DR, Bennett GN (1982) Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator. Gene 17:9–18
Shimada K, Weisberg RA, Gottesman ME (1972) Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J Mol Biol 63:483–503
Shimizu Y, Shimizu N, Hayashi M (1973) In vitro repression of transcription of the tryptophan operon by trp repressor. Proc Natl Acad Sci USA 70:1990–1994
Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Singleton CK, Roeder WD, Bogosian G, Somerville RL, Weith HL (1980) DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor. Nucl Acdis Res 8:1551–1560
Stormo GD, Schnieder TD, Gold LM (1982) Characterization of translational initiation sites in E. coli. Nucl Acids Res 10:2971–2990
Squires CL, Lee FD, Yanofsky C (1975) Interaction of the trp repressor and RNA polymerase with the trp operon. J Mol Biol 92:93–111
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
Vogel HJ, Bonner DM (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106
Yanofsky C (1981) Attenuation in the control of expression of bacterial operons. Nature 289:751–758
Yanofsky C, Platt T, Crawford IP, Nichols BP, Christie GE, Horowitz H, Van Cleemput M, Wu AM (1981) The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucl Acids Res 9:6647–6668
Zubay G, Morse DE, Schrenk WJ, Miller JHM (1972) Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli. Proc Natl Acad Sci USA 69:1100–1103
Zurawski G, Gunsalus RP, Brown KD, Yanofsky C (1981) Structure and regulation of aroH, the structural gene for the tryptophan repressible 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase of Escherichia coli. J Mol Biol 145:47–73
Author information
Authors and Affiliations
Additional information
Communicated by G. O'Donovan
Rights and permissions
About this article
Cite this article
Bogosian, G., Somerville, R.L. Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for Trp repressor. Molec. Gen. Genet. 193, 110–118 (1984). https://doi.org/10.1007/BF00327423
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00327423