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A Cit-Dependent Promoteris Located Within the Q Gene of Bacteriophage X

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A Cit-Dependent Promoteris Located Within the Q Gene of Bacteriophage X Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3134-3138, May 1985 Biochemistry A cIT-dependent promoter is located within the Q gene of bacteriophage X (transcriptional activation/promoter mutation/antisense RNA) BARBARA C. HOOPES AND WILLIAM R. MCCLURE Department of Biological Sciences, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213 Communicated by Allan Campbell, January 11, 1985 ABSTRACT We have found a eII-dependent promoter, nism of this inhibition is not completely understood. Under PaQ, within the Q gene of bacteriophage X. Transcription conditions where cIl is overproduced, such as in a cro- experiments and abortive initiation assays performed in vitro phage, cII-dependent inhibition has been shown to result in showed that the promoter strength and the clI affinity of PaQ severe growth defects for the phage (9, 10). Court et al. (11) were comparable to the other cU-dependent X promoters, PE extended the observations of McMacken et al. (8) to dem- and PI. The location and leftward direction of PaQ suggests a onstrate that a X cI-cy- phage showed a similar early possible role in the delay of X late-gene expression by clI appearance of late proteins as a X c-fcII- phage. They protein, a phenomenon that has been called cM-dependent concluded that cII-dependent inhibition of late protein syn- inhibition. We have constructed a promoter down mutation, thesis resulted from a decrease in Q gene expression through paq-l, by changing a single base pair in the putative clI binding inhibition ofPR transcription by the convergent PE promoter. site of the promoter by oligonucleotide site-directed Indeed, Schmeissner et al. (12) have found a 50% reduction mutagenesis. Thepaq-1 mutant promoter required about 4-fold in PR-specific RNA in a cII+ as opposed to cII- infection. It higher cdi concentrations for maximal activation compared to has been reported, however, that a PE mutation does not the wild-type PaQ. We tested the hypothesis that PaQ is entirely eliminate cII-dependent inhibition (9, 10, 13). The responsible in part for the delay of X late-gene expression by observation (D. Court, cited in ref. 14) that a sequence recombining the paq-1 mutation into a phage showing severe resembling PE exists within the Q gene suggested an ad- cU-dependent inhibition. We found that the paq-l mutation ditional contribution to cII-dependent inhibition. Transcrip- relieved the cIT-dependent growth defect of this phage. The tion from this leftward-transcribing promoter could inhibit paq-1 mutation (in combination with X c1857) resulted in a the synthesis ofthe Q protein, the antiterminator required for clear-plaque phenotype at the permissive temperature of 320C. late gene expression (15), through transcriptional or transla- The role of the PaQ-initiated antisense transcript in the control tional interference. of X development is discussed. We have found that the DNA sequence noted by Court does indeed define a cIT-activated promoter which we call The clI protein of phage X has been called the "critical PaQ (anti-Q promoter). We have tested the hypothesis that switch" in the lysis/lysogeny decision for the phage (1). clI PaQ is responsible in part for cII-dependent inhibition by is required for activation of the PE and PI promoters, which constructing a promoter mutation, paq-1, and by recombin- are responsible for the transcription of the A cI (repressor) ing the mutated promoter into a X phage. We found that the and int (integrase) genes, respectively. Both of these gene recombination of the paq-J mutation with a cro- phage products are required for the establishment of lysogeny. In showing severe cII-dependent inhibition relieved the cII- addition, much of the input to the phage developmental dependent growth defect of the phage. We have also con- program by the host is mediated by effects on cII synthesis structed the X cI857 paq-J phage and found that it displayed or stability (for review, see ref. 2). The activation of PE and a clear-plaque morphology at 32°C. PI by cII has been extensively characterized. Both promoters require cII in addition to RNA polymerase for transcription MATERIALS AND METHODS in vitro and both show poor homologies to prokaryotic promoter consensus sequences (3). Chemical protection, Endonucleases [Ava TI, HindIII, and EcoRI (Bethesda Re- DNase I "footprinting" experiments (4) and in vivo (2) and in search Laboratories); Bgl IT, Rsa I, and Nru I (New England vitro (5, 6) analysis of mutations at the PE promoter have Biolabs); Cla I (Boehringer Mannheim)]; unlabeled shown that the clI site can be defined T-T-G-C- ribonucleoside (ICN) and deoxyribonucleoside triphosphates binding by (P-L Biochemicals); agarose (SeaKem Laboratories, N6-T-T-G-C, where N6 corresponds in position to the -35 Rockland, ME); [a-32P]GTP, [a-32P]ATP, and [a-32P]dATP region hexamer. Interaction of cII with this sequence results (Amersham); acrylamide and N,N'-methylenebisacrylamide in large increases in both the initial binding of RNA poly- (Bio-Rad); cytidylyl(3'-5')adenosine (CpA; Sigma); and merase at the promoter and its subsequent conversion to an 3MM paper (Whatman) were purchased from the sources active complex (ref. 7 and unpublished data). indicated. RNA polymerase was purified from Escherichia The cII protein has an additional role in A lytic develop- coli K-12 by the procedure of Burgess and Jendrisak (16) with ment that has remained obscure. clI acts to inhibit late gene the modifications of Lowe et al. (17); the concentration was expression, a phenomenon which has been called "clI- determined by absorbance at 280 nm, assuming an A 1% of6.2 dependent inhibition." McMacken et al. (8) first observed and Mr = 4.9 x 105. The activity of the RNA polymerase was that aA cII- or A cIII- phage produced late proteins and late 60%, determined as described by Cech and McClure (18). X mRNA earlier than a A cI- phage. They proposed that cIT c1I protein was isolated as described (19) from the over- protein directly inhibited late mRNA synthesis. The mecha- producing strain N6308(pOG7), which was obtained from M. Gottesman (National Institutes of Health) (20); cIT protein The publication costs of this article were defrayed in part by page charge concentration was determined by absorbance at 280 nm, payment. This article must therefore be hereby marked "advertisement" using a molar extinction coefficient of 7.2 x 104 (19), and is in accordance with 18 U.S.C. §1734 solely to indicate this fact. given as nM monomers. X cI protein was a gift of R. T. Sauer 3134 Downloaded by guest on September 30, 2021 Biochemistry: Hoopes and McClure Proc. Natl. Acad. Sci. USA 82 (1985) 3135 (Massachusetts Institute of Technology). The Klenow frag- consensus sequence in Q corresponded to a cII-dependent ment of DNA polymerase was a gift of W. Brown (Carnegie- promoter by in vitro transcription ofX DNA. Phage DNA was Mellon University); it was isolated from the overproducing digested with the restriction endonuclease Ava II to generate strain CJ155 (21) and had a specific activity of 13,000 a run-off transcript from PI (267 bases), Bgl II to generate a units/mg. T4 DNA ligase was isolated as described (22). The run-off transcript from PE (240 bases), and Cla I to generate oligonucleotide primer used for the mutagenesis was ob- a run-offtranscript from the putative PaQ promoter (about 320 tained from the DNA synthesis service at the University of bases). As shown in Fig. 1A, we observed two cII-dependent Pennsylvania. transcripts for each reaction, as well as the self-terminating Solutions. LB, H top agar, and H plates were as described cII-independent transcript from PR (198 bases). One of these by Miller (23). MS buffer is 10 mM Tris Cl, pH 7.5/10 mM cII-dependent transcripts was ofthe size predicted for run-off MgCl2/50 mM NaCl/1 mM dithiothreitol/bovine serum albu- transcription from PI, PE, and PaQ, respectively. The other min (100 gg/ml). Xdil is 10 mM Tris Cl, pH 7.5/10 mM was independent of the restriction endonuclease used and MgSO4. TBE is 90 mM Tris/90 mM, boric acid/2.5 mM was about 220 bases long. As shown in Fig. 1B, this EDTA, pH 8.3. TE is 10 mM Tris Cl, pH 8.0/1 mM EDTA. self-terminated 220-base transcript also originated at PaQ. Assay buffer is 40 mM Tris Cl, pH 8.0/100 mM KCl/10 mM When the plasmid pBH100, which contains an EcoRI frag- MgCl2/1 mM dithiothreitol/bovine serum albumin (100 ment containing the Q gene (see below), was digested with .Ag/ml). EcoRI and transcribed in the presence of cHI, both the Bacterial and Phage Strains and Plasmid Constructions. predicted run-off transcript (320 bases) and the 220-base GM33 [dam 4 (24); from M. Susskind (Univ. of Mas- terminated transcript were observed. The appearance ofboth sachusetts Medical School)], W1485F+:: TnlO-2 [E. coli of these transcripts was dependent upon the addition of clI, K-12 supE42/F+ zzz::TnlO-2 (25); R. Zagursky (National even after long preincubations of the DNA with RNA Cancer Institute)]; C600K- [supE; M. Rosenberg (Smith polymerase or at high RNA polymerase concentrations (data Kline & French Laboratories)]; and XK1500 [AlacUl69- not shown). All three cII-dependent promoters appeared to Sms B1; E. Minkley (Carnegie-Mellon Univ.)] were obtained be of equal strength in transcription experiments, and PaQ from the laboratories cited. GM33F+:: TnJO-2 was con- was active at the same low clI concentrations as were PE and structed for use as a dam-F+ strain.
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