Identification and Characterization of a New Transcriptional Termination

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7373-7377, December 1984 Biochemistry Identification and characterization of a new transcriptional termination factor from Escherichia coli (tau factor/T7 phage terminator/RNA polymerase) JEAN-FRANCOIS BRIAT AND MICHAEL J. CHAMBERLIN Department of Biochemistry, University of California, Berkeley, CA 94720 Communicated by H. A. Barker, August 9, 1984 ABSTRACT We have identified and partially purified an stem with a four-base loop (ref. 7; see below). Termination at activity from Escherichia coli that enhances transcription ter- this site is not affected by rho mutations (9) and is not com- mination at the bacteriophage T7 early terminator when pletely efficient in vivo (9) or in vitro (10). It has been found cloned on the plasmid pAR1707. The factor also causes the that about 50% of T7 RNA chains terminate in vitro at T7 transcript to be terminated at a site several nucleotides earlier nucleotide 7588 ending in a 3' OH cytidine, while the remain- than in its absence. The resulting 3' OH ends of the transcripts der terminate at the adjacent guanosine residue (11), al- are identical to those found in vivo by S1 nuclease mapping. though other sequences have been reported (12). We show From this we conclude that the factor we have identified is here that the efficiency of in vitro termination at Te is en- probably responsible for determination of the 3' OH ends of hanced by an E. coli protein factor that shifts the 3' OH ter- T7 RNA transcripts in vivo. This factor does not act by pro- minus four bases, to a site coincident with that found for cessing a preformed RNA transcript, nor is it replaced by rho termination in vivo. protein or nusA or nusB proteins. Therefore, it appears to be a new transcription termination factor, and we have designated it "tau factor." Elucidation of its role in transcription in E. MATERIALS AND METHODS coli will depend on its purification to homogeneity and further studies of its properties. Materials. Nucleoside triphosphates were purchased from P-L Biochemicals. [a-32P]CTP was prepared as described by Control of cell growth and gene expression in bacterial cells Symons (13). DEAE-cellulose (DE-52) was from Whatman. is achieved mainly at the level of transcription. While regula- Heparin-agarose was prepared as described by Davison et tion can take place at any step in the transcription process, al. (14). E. coli RNA polymerase holoenzyme was purified much attention has focused recently on the steps of elonga- as described by Burgess and Jendrisak (15) as modified by tion and termination (1-4). Regulation of these processes by Gonzales et al. (16). The plasmid pAR1707 (Fig. 1) was gen- attenuation and antitermination plays a major role in bacteri- erously provided by W. F. Studier (Brookhaven National al systems, yet many of the components and mechanisms are Laboratory). not yet well understood. pAR1707 DNA was prepared by using the boiling proce- Termination of bacterial transcription is believed to in- dure described by Holmes and Quigley (17) and purified by volve a specific signal that stops elongation by RNA poly- two successive CsCl gradient centrifugations. pAR1707 merase and then allows release of the nascent RNA chain. DNA was used as in vitro template after digestion with Sal I, The bacterial transcription termination signals that have heat inactivation of restriction endonuclease for 10 min at been studied thus far generally include a potential stem-loop 70'C, and precipitation with ethanol. Restriction enzyme-di- structure in the RNA just upstream of the 3' OH terminus of gested DNA was resuspended in 10 mM Tris HCl, pH 8.0/1 the completed RNA chain. They commonly have been clas- mM EDTA to a concentration of 1 mg/ml. sified as "rho independent" or "rho dependent," depending The probe for S1 nuclease mapping was prepared from a on whether efficient termination occurs in vitro with purified 448-bp BamHI-Sal I DNA fragment (see Fig. 1) purified by Escherichia coli RNA polymerase alone or only in the pres- polyacrylamide gel electrophoresis and eluted as described ence of the termination factor rho (1). This classification is by Maxam and Gilbert (18). The 3' end of the BamHI site not sharply drawn; some rho-independent termination sites was labeled by using [a-32P]dGTP (3000 Ci/mmol, ICN; 1 Ci are enhanced when rho protein is added in vitro (5) and ter- = 37 GBq) and the large Klenow fragment (New England mination in vivo is reduced by rho mutations at some sites Biolabs) following the procedure of Smith et al. (19). Strand that do not respond to rho protein in vitro (6). In addition, it separation and sequencing of the probe were carried out as appears that termination at some sites requires the nusA pro- described by Maxam and Gilbert (18). tein (7), which may play a role in both elongation and termi- RNA Preparation and Assay of tau Factor Activity. In vitro nation (8). Finally, there is at least one site-the T3 phage RNA synthesis was carried out in a reaction mixture (50 Al) early terminator-that is highly efficient in vivo, but not in containing 40 mM Tris-HCl (pH 8.0); 10 mM 2-mercapto- vitro, and that is unaffected by rho mutations (9) or by nusA ethanol; 10 mM MgCI2; bovine serum albumin at 0.1 mg/ml; protein (unpublished observations), which has led to the sug- 0.4 mM each of GTP, UTP, ATP, and [a-32P]CTP (500-1000 gestion that other termination factors may exist. cpm/pmol); E. coli RNA polymerase holoenzyme at 40 One of the most studied rho-independent terminators is gg/ml; and Sal I-digested pAR1707 DNA at 20 ,ug/ml. When the early terminator (Te) of phage T7. This site maps at about appropriate, extract was added at a final concentration of 1.8 19% on the phage genome and consists of an 8-base-pair (bp) mg/ml to replace the purified RNA polymerase. The assay for termination factor activity during its partial purification The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: bp, base pair(s); nt, nucleotide(s); PA, and Te, early in accordance with 18 U.S.C. §1734 solely to indicate this fact. promoter Al and early terminator of phage T7. 7373 Downloaded by guest on September 27, 2021 7374 Biochemistry: Briat and Chamberlin Proc. NatL Acad ScL USA 81 (1984) was carried out by adding 4 pl of each fraction to a 25-jil orl reaction mixture containing purified E. coli RNA polymerase as described above. After 10 min at 370C, heparin was added (final concentration, 100 pug/ml) to block reinitiation of RNA chains, and incubation was continued for 10 min at 370C to allow full runoff of elongating transcripts. Then RNA transcripts were extracted with phenol as described by Kingston and Chamberlin (20) except that stop mix was added prior to phenol extraction. Analysis of RNAs was performed on 5% polyacrylamide/7 M urea gels or on high-reso- lution 8% polyacrylamide/7 M urea gels as described by Kingston and Chamberlin (20). Gels were dried and autora- diographed with Kodak XAR5 films. In vivo RNAs were prepared as described by Gilman and Chamberlin (21). The assay we have used for tau factor is only semiquanti- tative. As increasing amounts of partially purified factor are added to the reaction, there is first a reduction in the size of the 160-nucleotide (nt) terminated transcript, followed at higher concentrations by a progressive reduction in the yield Sall HI of read-through transcript. We have used the latter effect to measure activity, since it is difficult to measure the amount of each of the shorter transcripts. A "unit" of tau activity is that amount needed to reduce the yield of 502-nt read- through transcript by about 50%. In some cases we simply estimate tau activity by the percentage yield of read-through transcript, which varies from 35% (no tau) to 0% (excess FIG. 1. Transcription map of pAR1707. pAR1707 was construct- tau). ed by ligation of a 286-bp HindII fragment and of a 142-bp Fnu IV Protein concentrations were determined by using the Hi fragment from T7 DNA containing, respectively, the Al promot- Bradford method (22) with bovine serum albumin as stan- er PA, and the early terminator Te in the BamHI (HI) site of pBR322 dard. by using a BamHI linker (24). Black segments of map are T7 se- S1 Nuclease Mapping. S1 nuclease mapping experiments quences; white and shaded segments are pBR322 and linker se- were performed as described by Gilman and Chamberlin quences. Sizes (160 and 502) are shown in nt. (21). Preparation of Cell Extracts. E. coli fraction 1 extracts (su- Transcription of DNA from this plasmid in vitro by purified pernatants at 200,000 x g) were prepared from the following E. coli RNA polymerase holoenzyme gave predominantly a E. coli strains: DG156 (RNaseI-), BL107 (RNaseI-, RN- mixture of 160- and 161-nt transcripts, read from PA1 to Te, aseIII-), W3110 psu+ (lacZ4118, trpR, trpE4829, trpA4761), together with some read-through (Fig. 2, track H). This read- W3110 psu4; a procedure similar to that described by Fuller through could be visualized as a 502-nt transcript when the et al. (23) as modified by Reynolds and Chamberlin (unpub- DNA was cleaved with restriction endopuclease Sal I before lished data) was used. transcription (Fig. 2). About 35% read-through was found in Fractionation of tau Factor.

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