JOURNAL OF BACTERIOLOGY, OCt. 1982, p. 363-371 Vol. 152, No. 1 0021-9193/82/100363-09$02.0O/0 Copyright 0 1982, American Society for Microbiology Attenuation Regulation in the thr Operon of Escherichia coli K-12: Molecular Cloning and Transcription of the Controlling Region STEVEN P. LYNN,1 JEFFREY F. GARDNER,'* AND WILLIAM S. REZNIKOFF2 Department of Microbiology, University ofIllinois, Urbana, Illinois 61801,1 and Department ofBiochemistry, University of Wisconsin, Madison, Wisconsin S37062 Received 9 March 1982/Accepted 17 June 1982 Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%o. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator. The threonine (thr) operon ofEscherichia coli at the thr attenuator is defective in these mutant specifies four of the five enzymatic activities strains (17; S. Lynn, C. Bauer, K. Chapman, necessary to synthesize threonine from aspartic and J. Gardner, manuscript in preparation). The acid (15, 51) (Fig. 1). The structural genes model for regulation of the thr operon proposes (thrABC) of the operon (51) map at minute zero that translation of the leader RNA is involved in on the standard linkage map (1). A regulatory regulation of transcription termination at the thr region, originally identified by cis-dominant con- attenuator (17, 53). stitutive mutations, maps adjacent to the thrA This report describes the molecular cloning of structural gene (19, 43, 51). We were interested the thr operon regulatory region and in vitro in genetic regulation of the thr operon since transcription experiments localizing the thr op- expression ofthe structural genes is multivalent- eron promoter and attenuator. The in vitro tran- ly regulated by the intracellular levels of both scription results show that transcripts approxi- threonine and isoleucine (15). mately 150 to 170 bases in length are initiated in DNA sequencing studies have revealed that the thr regulatory region and terminated at or the thr operon may be regulated by an attenua- near the attenuator. Evidence is also presented tion mechanism (17) similar to other amino acid which indicates that these transcripts are syn- biosynthetic operons (3, 12, 20, 24, 26, 27, 28, thesized in vivo. 29, 38, 39, 54). Structural features of the thr regulatory region which have been identified by MATERIALS AND METHODS DNA sequence analysis include a potential cod- Bacteria, bacteriophages, plasmids, and media. E. ing region for a "leader peptide" and a tran- coli K-12 strains C600 SF8 (thrB leu thi str hsr hsm scription termination site (attenuator) approxi- recB recC lop) (49) and MO (F-, isogenic with Hfr H) mately 30 base pairs (bp) preceding the thrA (18) were used throughout this study. gene. The leader is 21 amino A pthr spi and A pthr79-20 spi are recombinant putative peptide phages which carry the thr operon controlling ele- acids in length and contains eight threonine and ments and the thrA and B structural genes (18). A four isoleucine codons. The attenuator was iden- pthr79-20 spi carries the constitutive thr79-20 muta- tified by its homology with other transcription tion. Plasmids pBR322 (7) and pVH51 (22) were used terminators and by regulatory mutations which as cloning vehicles. result in constitutive synthesis of the thr operon M9, TYE, LB (37), and Thr (18) were used as base enzymes. Presumably transcription termination media. The common L-amino acids (40 ,ug/ml), deoxy- 363 364 LYNN, GARDNER, AND REZNIKOFF J. BACTERIOL. asportote semialdehyde homoserine homoserine threonine asportokinose I dehydrogenose dehydrogenose I kinose synthase (EC 2.7.2.4) (EC 1.2.1.11) (EC 1.1.1.3) (EC 2.7.1.39) (EC 2.9.9.2) asport ic __ -osportyl osportic homoserine homoserine threonine acid phosphote 8-semioldehyde 0-phosphote thrA asd MthrA thrB thrC II lysine methionine isoleucine FIG. 1. Biosynthetic pathway for L-threonine. Aspartate semialdehyde dehydrogenase (asd) is unlinked to the thr operon and is regulated by lysine, methionine, and threonine. cholate (0.5% wt/vol), ampicillin (50 ,ug/mi), and tetra- described previously (6, 45). RNA polymerase was cycline (20 ag/ml) were used when required. purified by the method of Lowe et al. (31) or pur- Phage growth, pamid purification, and r n chased from Bethesda Research Laboratories. fragment isolation. Bacteriophages were propagated in Chemicals. All chemicals were of reagent grade. a- MO cells and the DNA was isolated as described by 32P-labeled ribonucleoside triphosphates were pur- Gardner and Reznikoff (18). Plasmid DNA was isolat- chased from New England Nuclear Corp. or Amer- ed as described by Maquat and Reznikoff (33). sham Corp. 32Pi was purchased from New England Restriction fragment templates were isolated by the Nuclear. method of Maxam and Gilbert (35) and further purified Isolatin of thr RNA synteszed in vivo. Labeling of by DE52 chromatography as described by Maquat et cellular RNA with 32Pi (20 mCi) and RNA isolation al. (34). DNA samples were stored in DNA buffer (10 were performed as described by Squires et al. (47). mM Tris-hydrochloride [pH 7.91-0.1 mM EDTA) at Five-microgram samples each of BamHI-digested 40C. pBR322 and pSL108 DNAs were subjected to agarose In vitro Urnsciption conditions and DNA sequenc- gel (1% wt/vol) electrophoresis, transferred to nitro- ing. Reactions were incubated for 10 min at 37°C in 50 cellulose filters, and hybridized with 32P-labeled RNA ,ul of 20 mM Tris-hydrochloride (pH 7.9)-0.1 mM as described by Davis et al. (11). Elution ofRNA from EDTA-0.1 mM dithiothreitol-150 mM KC1-4 mM nitrocellulose filters was accomplished by adding 300 MgCI2 with 120 mM of the four unlabeled nucleoside ,Al of 10 mM Tris-hydrochloride (pH 7.9)-i mM EDTA triphosphates, 10 to 50 gCi of [a-32P]CTP or [a- and the nitrocellulose strip to a 1.5-mi siliconized 32P]UTP, and 1 to 2 gig of plasmid DNA (0.2 to 0.4 Eppendorf tube and heating to 95°C for 5 min. The pmol) or 2 to 5 pmol of restriction fiagment DNA (14). filter was removed, and the RNA was precipitated by Reactions were initiated by addition of2 gug (1 pmol) of addition of 25 gag of tRNA, 35 gul of 3 M sodium RNA polymerase. After 20 min, rifampin was added to acetate, and 1 ml of absolute ethanol and kept at 10 gig/ml, and the reactions were incubated for an -70°C for S min. The mixture was centrifuged at additional 10 min. tRNA (25 gag) was added, and the 10,000 x g for 5 min, and the RNA was vacuum dried samples were then phenol extracted once, and 200 gul and subjected to electrophoresis as described above. of 0.3 M sodium acetate was added to each tube. The Ligtion and transformation conditios. Ligation re- samples were then ethanol precipitated at -70°C for 5 actions (10 to 20 gAl) contained 66 mM Tris-hydrochlo- min, and the pellets were washed with 95% ethanol, ride (pH 7.9), 6.6 mM MgCl2, 66 ,uM ATP, 10 mM dried, and subjected to gel electrophoresis on 8% dithiothreitol, and 5 mM spermidine. In experiments acrylamide-8 M urea gels as described by Maxam and utilizing pVHS1 as the vector, ligation reactions were Gilbert (35). Quantitation of radioactivity in gel slices carried out at 15C ovemight with 0.1 to 0.2 U of T4 was done by Cerenkov radiation in a liquid scintilla- DNA ligase. In experiments utilizing pBR322 as the tion counter. DNA sequencing and 5' end labeling vector, ligation reactions were carried out at 4°C were as described by Maxam and Gilbert (35). [y- ovemight with 5 U (52) of T4 DNA ligase purified in 32P]ATP was prepared by the method of Johnson and our laboratory. Transformations, using SF8, were Walseth (23). carried out as described by Maquat and Reznikoff(33). Coddn El, r n endonucleases, enzymes, and After transformation, the mixture was added to 4 ml of gel eectrophoreis conditions. Colicin El was prepared LB broth, and the cells were grown with shaking for 90 as described by Maquat and Reznikoff (33). AluI, min at 37C before colicin El treatment or direct EcoRI, HaeIII, HhaI, HindII, HindIII, RsaI, and TaqI plating onto antibiotic selection plates. were prepared by standard methods (21, 32, 36, 40, 41, Constrction of recombinant pIds. pJG10 and 44, 46). BamHI, BstEII, ClaI, and Sail were pur- pJG39 contain the HaeIII 1,700-bp (HaeIII-1,700) re- chased from Bethesda Research Laboratories and striction frament from A pthr spi (18) inserted into the were used as described by the manufacturer. T4 DNA unique HindII site of pVHS1. Ligation mixtures con- ligase and T4 DNA polymerase were purchased from tained 0.2 gg of HindII-digested pVHS1 and 0.1 gg of New England Bio-Labs or purified in our laboratory purified HaeIlI-1,700 DNA from A pthr spi.