Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7373-7377, December 1984 Biochemistry

Identification and characterization of a new transcriptional termination factor from (tau factor/T7 phage /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 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 polymer- ase 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 add- ed prior to phenol extraction. Analysis of RNAs was per- formed 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. All procedures were carried such experiments; this amount is somewhat higher than the out at 0-40C. E. coli fraction 1 from DG156 (8.5 ml) was levels of read-through found with intact T7 DNA using the brought to 60% ammonium sulfate by addition of 12.75 ml of purified E. coli RNA polymerase (25). saturated ammonium sulfate solution (4.1 M at 250C, pH Surprisingly, transcription of pAR1707 DNA in E. coli cell 7.3). The precipitate was collected after 30 min by centrifu- extracts presented quite a different pattern. First, the gation. The pellet was dissolved in 4 ml of buffer A (10 mM amount of read-through was reduced >90%, as measured by Tris, pH 7.5/10 mM MgCl2/1 mM EDTA/7.5% glycerol/0.3 the decrease in the amount of the 502-nt transcript (Fig. 2, mM dithiothreitol/50 mM NaCl) and dialyzed overnight track E; Table 1). Second, the yield of terminated transcripts against 2 liters ofthis buffer. The sample was then chromato- was increased correspondingly up to 15-fold (Table 1). Final- graphed on a column of heparin-agarose (1.5-cm diameter by ly, the size of the terminated transcript was reduced slightly, 5.5-cm length) that had been equilibrated with buffer A. Af- as compared to the transcript obtained with purified E. coli ter loading, the column was washed with 45 ml of buffer A RNA polymerase (Fig. 2, tracks D, E, and G). and then eluted with a linear gradient (36-ml total volume) AMtered Termination at T7 Te in Cell Extracts Is Due to a from 50 mM to 1 M NaCl in buffer A. Eighteen fractions Novel Termination Factor, tau. These alterations in tran- were collected and dialyzed overnight against buffer A. Ac- scription pattern are not due to breakdown or processing of tive fractions (from about 0.5 to 0.6 M NaCl) were pooled either the 502- or 160-nt transcripts. When these transcripts and chromatographed on a DEAE column (1.5-cm diameter were prepared with E. coli RNA polymerase holoenzyme, by 3.5-cm length). After loading, the column was washed purified, and then incubated with the E. coli extract for 20 with 10 ml of buffer A and then eluted with a linear gradient min at 370C, there was no decrease in the size of either tran- (20 ml) from 50 mM to 1 M NaCl in buffer A. Fifteen frac- script and little overall breakdown (data not shown). In- tions were collected and dialyzed overnight against buffer A stead, it appears that there is a factor in the extracts that prior to assay. enhances transcriptional termination at Te and that shifts the termination site to a position several nucleotides closer to the promoter. We have designated this activity as tau factor. RESULTS The activity of tau factor in E. coli extracts was lost after Transcription of pAR1707 in Cell Extracts Leads to Altered heating at 100'C or after extraction with phenol. However, Termination at T7 Te. Plasmid pAR1707, constructed by Stu- the activity was otherwise quite stable, even when kept at dier and Rosenberg (24), contains the strong early promoter 25TC for 3 days (Fig. 2, track G). The activity was purified Al (PAl) from bacteriophage T7, cloned together with the T7 about 40-fold by ammonium sulfate fractionation, followed early terminator (Te) into the BamHI site of pBR322 (Fig. 1). by heparin-agarose chromatography and DEAE-cellulose Downloaded by guest on September 27, 2021 Biochemistry: Briat and Chamberlin Proc. Natl. Acad. Sci. USA 81 (1984) 7375

A B C D E F G H Table 2. Partial purification of tau factor origin-n Volume, Protein, Amount of Fraction ml mg read-through* 200,000 x g super- natant 9.3 140 10 4W1 I 60% ammonium sulfate precipitate 5.8 80 21 Heparin-agarose pool 8.2 4.6 15 502 DEAE-cellulose pool 2.8 3.4 18 (RT) _ Assays were carried out in a 25-,pJ transcription mixture contain- ing E. coli RNA polymerase holoenzyme (40 tg/ml), pAR1707 di- gested with Sal 1 (20 ,sg/ml), and 4 ,ul of each fraction. Since 4 .d of each fraction gave about 50% reduction in read-through, the concen- tration of tau activity is roughly the same in each of the four frac- tions (-250 units/ml), and the specific activity of the DEAE-cellu- lose pool is about 13 times that of the initial extract. *The amount of the read-through was determined by analysis of the transcripts on a 5% polyacrylamide/7 M urea gel and calculated from the amount of 502-nt transcript relative to the amount of 160- nt transcript. chromatography (Table 2 and Fig. 3). The extent of purifica- tion was estimated from the amount of protein in different fractions that showed tau factor activity and from the reduc- tion in the amount of read-through of Te, as judged by RNA

16060 ;f _- gels (Fig. 4). However, the assay is only semiquantitative, (Te ) = a and the degree of purification is only an estimate (see Mate- rials and Methods). Gel analysis of the fractions across the peaks of tau activity from both the heparin-agarose and DEAE columns showed a coincidence between the reduc- tion of read-through transcripts and the shortening of the Te- terminated transcript (see Fig. 4). Hence, it is likely that both activities are due to the same factor. (Note, however, that the shortening effect was already detected at concentra- tions of tau factor that did not greatly reduce read-through). Tau factor does not appear to be any of the proteins cur- FIG. 2. In vitro transcription of pAR1707 digested by Sal I using rently known to be involved in transcriptional termination. different protein fractions. Tracks: A, E. coli RNA polymerase ho- The activity of tau factor was unchanged in the E. coli rho loenzyme (40 ug/ml) mixed with (0.2 mg/ml) and nusA mutant (6) psu4 (Fig. 2, track D). Purified E. coli rho (26) and protein (0.04 mg/ml); B, E. coli RNA polymerase holoerzyme (40 nusA (27) proteins, alone or in combination, did not mimic Ag/ml) mixed with nusA protein (0.04 mg/ml); C, E. coli RNA poly- tau factor in its effect on T7 Te termination (Fig. 2, tracks C, merase holoenzyme mixed with rho factor (0.2 mg/ml); D, fraction 1 B, and A). In addition, partially purified tau factor was ac- from E. coli W3110 mutant psu4 (1.7 mg/ml); E, fraction 1 from E. tive in enhancing termination and shortening the transcript at coli W3110 psu+ (1.8 mg/ml); F, heparin-agarose pool fraction from E. coli DG156 (0.875 mg/ml); G, fraction 1 from E. coli DG156 after 17 Te, even when the normal triphosphate substrates were 3 days at 25°C (1.8 mg/ml); H, E. coli RNA polymerase holoenzyme replaced by the four f3,y-imido analogs (data not shown). (0.94 mg/ml). RNAs were prepared as described and analyzed on an Rho factor does not function with these substrates (28). Fur- 8% polyacrylamide/7 M urea gel (40 cm x 15 cm x 0.3 mm) until thermore, previous studies on rho termination have estab- xylene cyanol reached the bottom of the gel. RT, read-through; T, lished that it has no effect on RNA chains shorter than 300 nt terminated. Sizes are shown in nt. (4). We also tested purified nusB protein, and it had no effect

Table 1. Effect of different factors on the efficiency of transcription termination at T7 Te Transcription termination Protein Read- Relative increase concentration, through, in terminated Components mg/ml % transcript E. coli polymerase alone 35 1.0 E. coli psu+ extract 1.8 3 15 E. coli psu4 extract 1.7 2 19 E. coli polymerase + rho factor 0.2 37 1 + nusA protein 0.04 20 2 + rho factor/nusA protein 0.2 + 0.04 30 1 Transcription reactions (see Methods) contained 4 A.l of extracts or factors where indicated. After transcription, samples (i04 cpm) were loaded on a 5% polyacrylanqide/7 M urea gel and separated by electrophoresis. Gels were analyzed by autoradiography, and slices were analyzed to determine the amount of label associated with the 160- and 502-nt regions. These amounts were used to calculate the percentage of read-through. The increase in termination was calculated relative to RNA polymerase alone as 1.0. Downloaded by guest on September 27, 2021 7376 Biochemistry: Briat and Chamberlin Proc. NatL Acad Sci. USA 81 (1984)

A B C D E F G H I J K

3

2 00 5 i2 I- - (R T)

z 0

1

Fractions 160 6 A a 1 '0 also$~~~~.Ex 0Te*)( 40 1.5

30 1 EX - r_ &- 20 z 0._ 0.5 10.

FIG. 4. In vitro transcription of pAR1707 digested by Sal I using E. coli RNA polymerase holoenzyme (track A) in the presence of 4 2 4 6 8 10 12 14 1l of the pool of heparin agarose fractions (fractions 11-15) (track Fractions B), or in presence of 4 gl of the following DEAE-cellulose fractions: 3, 4, 5, 6, 7, 8, 10, 12, and 14 (tracks C through K). Sizes are shown FIG. 3. Heparin-agarose chromatography (A) and DEAE-cellu- in nt. lose chromatography (B) of tau factor. o, % of 502-nt read-through (RT) transcript; *, protein concentration; ---, [NaCI]. quences as those seen in vivo. Again, there was no evidence the 3'-terminal se- on Te termination (data not shown). From these observations of exonucleolytic shortening of longer we conclude that tau factor is not rho, nusA, or nusB pro- quences by incubation with either extracts or tau factor tein. preparations in vitro (Fig. SA, tracks G and H). Termination at T7 Te in Vivo Takes Place at the Same Sites Found with tau Factor in Vitro. If tau factor is involved in DISCUSSION termination at the T7 Te in vivo, then this role should be evi- The early terminator of bacteriophage T7 shares several fea- dent from the 3' OH sequence of the transcript. We mapped tures in common with other rho-independent termination these sequences using the S1 nuclease mapping procedure sites (for review, see ref. 2). The site is somewhat unusual in (21) with a 3'-labeled, single-stranded probe. The probe was that it does not have the long, uninterrupted string of thymi- labeled at the BamHI site between PA, and Te and extended dine residues that is characteristic of many other rho-inde- to the Sal I site (Fig. 1). Hybridization of probe to pAR1707 pendent termination sites. Termination at T7 Te occurs with transcripts gave full-length protected probes from the 502-nt comparable efficiency in vivo (29) and in vitro (25). Howev- read-through transcripts and a set of protected fragments of er, the terminator for the closely related phage T3 is only about 100 nt from transcripts terminated near T, (Fig. SA). effective in vivo. Since rho mutations do not alter the effi- These could be indexed to a DNA sequencing gel prepared ciency of termination at T, for either phages T7 or T3, it has with the same probe. It was found that termination in vitro been suggested that other bacterial termination factors may with purified RNA polymerase was primarily at the cytidine be present in vivo (9). In addition, it is not implausible that shown in Fig. SB (T7 map position 7588) with some termina- termination at rho-independent terminators in vivo may in- tion at the adjacent guanosine, as reported by others (11). volve factors that enhance the termination and release Transcripts found in vivo, in contrast, were three or four phases of transcription, even though these factors are not bases shorter, ending primarily with uridine (7584) or cyti- essential for termination in vitro. dine (7585), although there were traces of termination at the We have identified and partially purified an activity from adjacent base as well. Transcripts synthesized in vitro in ex- E. coli that enhances transcription termination at phage T7 tracts (Fig. 5A, track E) or with a partially purified tau factor T, when cloned into the plasmid pAR1707. The factor also preparation (Fig. 5A, track F) gave the same 3' terminal se- causes the transcript to be terminated at a site several nucle- Downloaded by guest on September 27, 2021 Biochemistry: Briat, and Chamberlin Proc. Natl. Acad. Sci. USA 81 (1984) 7377

A A B C D E F G H otides earlier than in its absence. The resulting 3' OH ends of the transcripts are identical to those found in vivo by S1 nu- clease mapping. From this we conclude that the factor we have identified is probably responsible for determination of the 3' OH ends of T7 RNA transcripts in vivo. !tr This factor does not act by processing a preformed RNA transcript nor is it replaced by rho protein or nusA or nusB proteins. Therefore, it appears to be a new transcription ter- mination factor, and we have designated it as tau factor. Elu- cidation of its role in transcription in E. coli will depend on its purification to homogeneity and further studies of its properties. A A These studies were supported by a research grant (GM12010) A from the National Institute of General Medical Sciences. J.-F.B. is a postdoctoral fellow supported by the Centre National de la Re- C cherche Scientifique (France) and is the recipient of a North Atlan- G tic Treaty Organization grant. C a~~~~ A G 1. Roberts, J. W. (1976) in RNA Polymerase, eds. Losick, R. & A Chamberlin, M. J. (Cold Spring Harbor Laboratory, Cold A Spring Harbor, NY), pp. 247-271. A -3 2. Rosenberg, M. & Court, D. (1979) Annu. Rev. Genet. 13, 319- G 0-SI. -@ 343. G 3. Adhya, S. & Gottesman, M. (1978) Annu. Rev. Biochem. 47,

3- 967-996. 4. von Hippel, P. H., Bear, D. G., Morgan, W. D. & McSwig- gen, J. A. (1984) Annu. Rev. Biochem. 53, 389-446. 5. Howard, B., de Crombrugghe, B. & Rosenberg, M. (1977) Nu- cleic Acids Res. 4, 827-842. 6. Korn, L. J. & Yanofsky, C. (1976) J. Mol. Biol. 106, 231-241. 7. Platt, T. (1981) Cell 24, 10-23. 8. Kassavetis, G. A. & Chamberlin, M. J. (1981) J. Biol. Chem. 256, 2777-2786. 9. Kiefer, M., Neff, N. & Chamberlin, M. J. (1977) J. Virol. 22, 548-552. B UC 10. Millette, R. L., Trotter, C. D., Herrlich, P. & Schweiger, M. U G C-G (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 135-142. C-G 11. Dunn, J. J. & Studier, F. W. (1980) Nucleic Acids Res. 8, A-U 2119-2132. C-G UG In vitro termination sites 12. Peters, G. G. & Hayward, R. S. (1974) Eur. J. Biochem. 48, C-G 199-208. 13. Symons, R. H. (1977) Nucleic Acids Res. 4, 4397-4355. 5' pAUGUAAUCACACUGCUUUCUGCGUUUAU 3' 14. Davison, B. L., Leighton, T. & Rabinowitz, J. C. (1979) J. ft Biol. Chem. 254, 9200-9226. in vivo termination sites 15. Burgess, R. R. & Jendrisak, J. J. (1975) Biochemistry 14, 4634-4642. FIG. 5. Analysis of in vivo and in vitro termination sites of tran- 16. Gonzales, N., Wiggs, J. & Chamberlin, M. J. (1977) Arch. Bio- scripts ending at Te by S1 nuclease mapping. (A) 3' nuclease S1 map- chem. Biophys. 182, 404-408. ping of the RNA synthesized from pAR1707. A 448-bp BamHI-Sal I 17. Holmes, D. J. & Quigley, M. (1981) Anal. Biochem. 114, 193- fragment from pAR1707 was labeled at the 3' end of the BamHI site 197. by using [32P]dGTP and the Klenow fragment of E. coli DNA poly- 18. Maxam, A. H. & Gilbert, W. (1980) Methods Enzymol. 65, merase. The 3'-end-labeled strand was separated, sequenced, and 499-560. used as a probe for S1 mapping. Tracks: A, G+A; B, T+C; C, S1 19. Smith, M., Leung, D. W., Gillam, S., Astell, C. R., Montgom- mapping of in vivo RNAs (20 ,ug) purified from E. coli cells contain- ery, D. L. & Hall, B. D. (1979) Cell 16, 753-761. ing pAR1707; D, S1 mapping of in vitro RNAs synthesized from R. E. M. J. (1981) Cell 27, 523-531. pAR1707 digested by Sal I using E. coli RNA polymerase holoen- 20. Kingston, & Chamberlin, zyme; E, in vitro RNAs obtained with fraction 1 from DG156 (1.8 21. Gilman, M. Z. & Chamberlin, M. J. (1983) Cell 35, 285-293. mg/ml); F, analysis of in vitro RNA synthesized in the presence of 22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. the heparin-agarose fraction containing tau factor (0.875 mg/ml); G 23. Fuller, R. S., Kaguni, J. M. & Komberg, A. (1981) Proc. Natl. and H, in vitro RNAs obtained with E. coli RNA polymerase holo- Acad. Sci. USA 78, 7370-7374. enzyme and purified prior to incubation for 20 min at 370C, respec- 24. Studier, F. W. & Rosenberg, A. H. (1981) J. Mol. Biol. 153, tively, with the fraction 1 from DG156 (1.8 mg/ml) or with the hepa- 503-527. rin-agarose fraction containing tau activity (0.875 mg/ml). Hybrid- 25. Chamberlin, M. J., Nierman, W. E., Wiggs, J. L. & Neff, N. izations were performed in a 10-1.l volume for 3 hr at 660C. Nuclease (1979) J. Biol. Chem. 254, 10061-10069. S1 digestions were carried out with 2000 units of enzyme per sample 26. Roberts, J. W. (1%9) Nature (London) 224, 1168-1174. for 30 min at 370C. Samples were analyzed on a 6% polyacryla- mide/8.3 M urea gel (40 cm x 15 cm x 0.3 mm) as described by 27. Schmidt, M. C. & Chamberlin, M. J. (1984) Biochemistry 23, Maxam and Gilbert (18). (B) RNA sequence and secondary structure 197-203. Te from phage T7 DNA as described by Dunn and Studier (11). Low- 28. Gallupi, G., Lowery, C. & Richardson, J. P. (1976) in RNA er arrows indicate in vivo termination sites. Upper arrows show in Polymerase, eds. Losick, R. & Chamberlin, M. (Cold Spring vitro termination sites obtained with E. coli RNA polymerase holo- Harbor Laboratory, Cold Spring Harbor, NY), pp. 657-665. enzyme without tau factor added. 29. Studier, F. W. (1972) Science 176, 367-376. Downloaded by guest on September 27, 2021