THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistq and Moleculal ’ Biology, Inc.

Vol. 267, No. 21, Issue of September 25, pp. 19418-19426,1992 Printed in U.S.A.

Host Factor Requirements for Processive Antitermination of Transcription and Suppression of Pausing by the N Protein of Bacteriophage X*

(Received for publication, April 21, 1992)

Stephen W. Mason, Joyce Li, and Jack GreenblattS From the Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5G 1 L 6 Canada

The N protein of phage X prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the X early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each X early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the X terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermina- tion at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reac- tion. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N- modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the X nut sites. This substantial proc- essivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the X chromosome.

The N protein of bacteriophage X regulates transcription elongation by modifying Escherichia coli RNA polymerase so that termination signals are no longer recognized (Friedman et al., 1973; Adhya et al., 1974; Franklin, 1974; reviewed in Friedman, 1988; Roberts, 1988). This process, called antiter- mination, requires the nut (N-utilization) sites which are present in the two phage operons, p L and pR, that are regu- lated by the N protein (Salstrom and Szybalski, 1978; Olsen et al., 1982). The functional form of the nut site is in the nascent RNA transcript (Nodwell and Greenblatt, 1991).

Several E. coli proteins are also involved in antitermination by the N protein: NusA (Friedman and Baron, 1974), NusB (Keppel et al., 1974; Friedman et al., 1976), ribosomal protein S10 (Friedman et al., 1981; Das et al., 1985), and NusG

* This work was supported by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 416- 978-5863; Fax: 416-978-8528.

(Downing et al., 1990; Sullivan et al., 1992; Li et al., 1992). In reactions containing only E. coli RNA polymerase, termina- tion factor Rho, and the host proteins NusA, NusB, S10, and NusG, N causes a net increase in transcription of a circular DNA template containing a nutR site (Li et al., 1992). There- fore, it seemed likely that transcript elongation in vitro by RNA polymerase could extend over great distances in the presence of N and all four host factors.

The host proteins NusA, NusB, S10, and NusG all stably associate with transcription elongation complexes containing the N protein (Barik et al., 1987; Horwitz et al., 1987; Mason and Greenblatt, 1991) and are all required to stably bind nut site RNA in vitro (Nodwell and Greenblatt, 1991). The assem- bly of this ribonucleoprotein complex is guided by the surface of the elongating RNA polymerase (Mason and Greenblatt, 1991; Nodwell and Greenblatt, 1991). Complex formation is highly cooperative and involves multiple protein-protein and protein-RNA interactions (Mason and Greenblatt, 1991; Nod- well and Greenblatt, 1991). Interactions between N and NusA (Greenblatt and Li, 1981a), NusA and RNA polymerase (Greenblatt and Li, 1981b), S10 and RNA polymerase (Mason and Greenblatt, 1991), NusB and S10 (Mason et al., 1992), and NusG and RNA polymerase (Li et al., 1992) have all been demonstrated in vitro. These protein-protein and protein- RNA interactions probably all contribute to the overall sta- bility of the N-modified transcription complex.

It has been shown previously that N and NusA are sufficient for antitermination i n vitro at a Rho-independent terminator cloned downstream from the X nutL site (Whalen et al., 1988; Whalen and Das, 1990). However, transcription elongation from the X early promoter, pR, stimulated by N, can persist over distances as great as 10-20 kb’ and allow N-dependent transcription of the X late genes (Dambly and Couturier, 1971; Greenblatt, 1972). This highly processive antitermination by N protein requires NusA, NusB, and S10, since mutations in the genes coding for these factors impair antitermination both i n vivo and in vitro (Friedman and Baron, 1974; Keppel et al., 1974; Friedman et al., 1976; Friedman et al., 1981; Das and Wolska, 1984; Das et al., 1985; Goda and Greenblatt, 1985).

In this paper we examine the roles of the individual host factors NusA, NusB, S10, and NusG in the efficiency and processivity of antitermination by N i n uitro. We show that N and NusA are sufficient to prevent both pausing by RNA polymerase and termination of transcription over a short distance at the Rho-dependent X terminator, tR1 (Roberts, 1969; Rosenberg et al., 1978). However, N and NusA are not sufficient for efficient antitermination over distances much greater than 100-200 base pairs. The presence of all four host

The abbreviations used are: kb, kilobase(s); SDS, sodium dodecyl sulfate.

19418

Host Factors and Antitermination by X N Protein 19419

elongation factors, NusA, NusB, S10, and NusG, allowed antitermination by N to be much more processive and extend over at least 7000 base pairs in vitro.

EXPERIMENTAL PROCEDURES

Materials-RNA polymerase was purified as described by Burgess and Jendrisak (1975) or purchased from Boehringer Mannheim. N, NusA, NusB, and NusG were purified as described previously (Green- blatt et al., 1980; Greenblatt and Li, 1981b; Swindle et al., 1988; Li et al., 1992). Purified S I0 was the generous gift of V. Nowotny. Purified XcI repressor protein was the generous gift of A. Astromoff and M. Ptashne, Harvard University. The plasmids pLSl and pLSll (Lau and Roberts, 1985) were prepared by CsCl gradient centrifugation (Maniatis et al., 1982). Bacteriophage X DNA was purified by CsCl gradient centrifugation of phage particles followed by phenol/chlo- roform extraction of the DNA.

Restriction enzyme fragments of X DNA that were used for hybrid- ization are listed in Table I according to the standard X coordinates (Daniels et al., 1983). They were isolated by the crush and soak method (Maniatis et al., 1982) from polyacrylamide gels. These DNA fragments were either blotted directly onto nylon filters (ICN) or were cloned into the polylinker of the plasmid pTZ19R (Pharmacia LKB Biotechnology Inc.), and the uncut plasmids were blotted onto nylon filters (see below). Fragments for cloning were treated with E. coli DNA polymerase I Klenow fragment to produce blunt ends, followed by ligation with SmaI cut pTZ19R. Plasmid DNA was purified by CsCl gradient centrifugation (Maniatis et al., 1982).

Run-off Transcription Reactions-Transcription reactions (20 p1) were programmed with 30 nM of either the HinfI DNA fragment from pLS1 or pLS11 or the HpII fragment from pLS1. Reactions con- tained RNA polymerase (40 nM), NusA (55 nM), and where indicated: NusB (100 nM), S10 (175 nM), Nu& (100 nM), Rho (45 nM), and various concentrations of N. The reaction buffer contained either: 20 mM Tris-HCI, pH 7.9, 50 mM potassium chloride, 5 mM magnesium chloride, 6 mM 2-mercaptoethanol, 0.1 mM EDTA, 5% glycerol (Figs. 2 and 3) or 20 mM Tris acetate, pH 7.9, 150 mM potassium glutamate, 4 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM dithiothreitol (Fig. 4). The potassium glutamate buffer conditions have been shown to increase the activity of termination factor Rho (Zou and Richardson, 1991). All reactions also contained 0.2 mM each of ATP, CTP, and UTP, 0.04 mM GTP, 5 pCi of [w~'P]GTP, and 40 units of RNase inhibitor (Pharmacia). Transcription reactions were preincubated for 10 min a t 37 "C in the absence of unlabeled GTP and magnesium+. Transcription was initiated by the addition of magnesium+, GTP, and 10 pg/ml rifampicin, and the reactions were incubated for 15 min at 37 "C. Reactions were stopped by the addition of 80 pl of 30 mM EDTA, 25 pg/ml yeast tRNA and extracted sequentially with phenol/ chloroform and chloroform. Nucleic acids were precipitated with ethanol. The pellet was dried, boiled in formamide loading buffer, and electrophoresed on a 4% polyacrylamide gel in the presence of 8 M urea (Maniatis et al., 1982).

Transcription Reactions Analyzed by Slot Blot Hybridization-All transcription reactions for these experiments were prepared with buffers containing potassium glutamate, as described above. Tran- scription reactions (100 pl) were programmed with 0.6 nM X DNA and contained 25 pCi of [a-"PIUTP (instead of GTP), 0.2 mM each of ATP, CTP, and GTP, 0.04 mM UTP, and the various proteins indicated in the figure legends (60 nM RNA polymerase, 220 nM NusA, 210 nM NusB, 185 nM S10, 220 nM Nu&, 200 nM Rho, and 80 nM N, or as indicated). Reactions were incubated for 15 min at 37 "C followed by the addition of 25 units of DNase I (Pharmacia) and further incubation for 10 min at 37 "C. Reactions were stopped with the addition of 50 pl of 50 mM EDTA, 0.5% SDS, extracted with phenol/chloroform (l:l), and precipitated with ethanol. Dried pellets were resuspended in 200 pl of 50% formamide, 8.25 mg/ml yeast tRNA, 3.5 mg/ml salmon sperm DNA.

Fragments of A DNA or plasmid DNAs containing X DNA inserts (400 pmol) were denatured by the addition of one volume 0.8 N sodium hydroxide, incubated a t room temperature for 10 min, neu- tralized by the addition of one volume of 2 M ammonium acetate, pH 7, and left on ice for a t least 5 min. The DNA was blotted onto ICN nylon membrane by the addition of 400 pl of this solution to each slot of a Bio-Rad Bio-Dot apparatus. Each slot was washed with 500 pl of 2 X SSC (1 X ssc contains 15 mM sodium citrate, 150 mM sodium chloride), and the DNA was covalently attached to the mem- brane by exposure to an ultraviolet light source. The membranes were incubated in prehybridization solution (50% formamide, 7% SDS, 50

mM sodium phosphate, pH 6.8, 0.04 mM EDTA, 150 pg/ml yeast tRNA, 320 pg/ml denatured salmon sperm DNA) at 50 "C for 4-6 h. Labeled RNA probe was added to the filter in hybridization solution (same as prehybridization solution without tRNA and salmon sperm DNA), and incubation was continued a t 50 "C for approximately 16 h. The nylon filters were washed at room temperature successively in solutions containing: 2 X SSC; 2 x SSC, 0.1% SDS; 0.5 X SSC, 0.1% SDS; 0.1 X SSC, 0.1% SDS. The filters were air-dried and exposed to film.

RESULTS

N and NwA Are Sufficient to Prevent Termination at Proximal Sites in the Rho-dependent Terminator tR1-It has been shown previously that N and NusA are sufficient to prevent termination at the simple Rho-independent X termi- nator tR' (Whalen et al., 1988; Whalen and Das, 1990). We wanted to determine whether N and NusA are also sufficient for antitermination at the Rho-dependent X terminator tR1. Therefore, we performed run-off transcription assays using, as DNA template, a HinfI fragment from either a nut+ plasmid (pLS1 containing the pR-cro-nutR-tR1 region from the X rightward operon) or a nut- derivative of pLSl (pLS11; Lau and Roberts, 1985), as diagrammed in Fig. 1. Transcripts were labeled with [32P]GTP in reactions containing purified RNA polymerase, termination factor Rho, and NusA, either in the presence or absence of N. The reaction products were sub- jected to electrophoresis and detected by autoradiography (Fig. 2). In reactions that lacked N, transcription on either template was partially terminated at three sites (I, 11, and 111; Lau et al., 1982) within the tR1 terminator region (Fig. 2, lanes 1 and 3 ) . When the template was derived from pLSl l (Fig. 2, lanes 3 and 4 ) , there was less termination because the deletion in pLSl l removes rutA, a site that overlaps the nutR site and improves the efficiency of termination by Rho (Chen et al., 1986; Chen and Richardson, 1987). The addition of N to the reaction containing the nut+ DNA template allowed efficient read-through of sites I and I1 and reduced termina- tion at site 111, thus increasing the amount of run-off tran- script (Fig. 2, lane 2 ) . In contrast, N was unable to antiter- minate transcription at any of sites I, 11, or 111 on the nut- DNA template (Fig. 2, compare lanes 3 and 4). This ability of N to suppress termination at tR1 depended on NusA (data not shown). Therefore N and NusA were sufficient for the nut site-dependent suppression of transcription termination at the Rho-dependent terminator tR1.

N and NusA Prevent Pausing by RNA Polymerase-Since N and NusA are sufficient to prevent termination at both simple (Whalen et al., 1988) and Rho-dependent (Fig. 2) terminators, we wondered whether N might also be able to suppress pausing by RNA polymerase. This may be an im- + + N

x37w*x pR nuIR i38754

".) .N

Hps I1 DNA lraprnal H ld I DNA lrqrnsnl

C G C T C I T A C A C A ~ C C A E E G ~ G A A A A ~ P L S ~ ( W + I - D I A b o x 8

PLSl1 (""I.)

FIG. 1. Plasmids used in run-off transcription assays. The pR-cro-nutR-tR1 region of the X rightward operon showing the rela- tive lengths of run-off transcripts produced in the presence and absence of N (top arrows) on the HinfI and H p d DNA fragments used as templates in run-off transcription experiments. The sequence of the nutR site and the extent of the pLSll linker insertion/deletion (hatched box; Lau and Roberts, 1985) within nutR are also shown.

19420 Host Factors and Antitermination by X N Protein

portant mechanism of antitermination, since pausing is be- lieved to be important for termination by RNA polymerase (reviewed by: Yager and Von Hippel, 1987; Richardson, 1991). In addition, the ability of X Q protein, along with NusA, to prevent pausing by RNA polymerase at many sites, including Rho-dependent terminators (Grayhack et al., 1985; Yang and Roberts 1989), is thought to be central to its role as an antitermination factor. To test whether N could prevent pausing at the Rho-dependent X terminator tR1, we performed the experiments shown in Fig. 3. Transcriptional pausing a t tRI can be examined by taking advantage of short pauses by RNA polymerase that occur a t sites I, 11, and I11 within the

RO - 111 - II - I-

- RO - 111 - II “I

1 2 3 4 FIG. 2. NusA and a nut site are sufficient for antitermina-

tion by N at sites I, 11, and I11 of the tRZ terminator. Transcripts produced in reactions containing purified RNA polymerase, Rho factor, and NusA and programmed with the Hinfl DNA fragment from either pLSl (nutl?’; lanes I and 2) or pLSl l (nutR-; lanes 3 and 4 ) were resolved on a urea-polyacrylamide gel and visualized by autoradiography. Reactions 2 and 4 contained N, as indicated. The positions of transcripts terminated a t tRI sites I, 11, and I11 and the run-off transcripts ( K O ) on both templates are indicated. The differ- ences in transcript lengths between the two templates reflect the presence of different linker insertion mutations on the two plasmids (Lau and Roberts, 1985).

A

tR1 terminator region in the absence of Rho factor (Lau et al., 1983). Transcription reactions containing NusA, but no Rho factor, were programmed with HinfI DNA fragments from either a nut+ or nut- plasmid (see Fig. 1). The drug rifampicin was used to synchronize initiation by RNA polym- erase and to prevent reinitiation (see “Experimental Proce- dures”). Following initiation of transcription, the reactions were incubated for various short periods of time in order to observe pausing a t sites I, 11, and I11 of tR1. When the reaction was programmed with the HinfI fragment from pLSl and contained NusA, RNA polymerase paused a t three sites within the tR1 terminator region (Fig. 3A, lanes 1-6). These paused transcripts had the same gel mobilities as transcripts produced in a reaction that contained Rho (Fig.3A, lane 7) and, there- fore, corresponded to the tR1 termination sites I, 11, and 111. The presence of N, in addition to NusA, caused RNA polym- erase to read through tR1 with little detectable pausing (Fig. 3B, lanes 1-6). Thus RNA polymerase reached the run-off point sooner and some run-off transcripts were observed even at the earliest time point (Fig. 3B, lane 1) . When the HinfI fragment from the nut- plasmid was used, the RNA polym- erase paused a t all three sites in tR1, even in the presence of N (Fig. 3C, lanes 1-6). Therefore, N and NusA are sufficient for nut site-dependent suppression of pausing by RNA polym- erase at the Rho-dependent terminator tR1.

NUB, 5’10, and NusG Are Important for Antitermination by N at More Distal Sites in tR1-In order to assess the effect of the additional host elongation factors NusB, S10, and NusG on antitermination by N and NusA a t tR1, we performed run- off transcription assays containing various concentrations of X N protein, either in the presence of NusA alone or in the presence of all four host factors. Since NusA alone was sufficient for antitermination by N on the short HinfI frag- ment containing sites I, 11, and I11 of tR1 (Fig. 2), these reactions contained a template with additional sequences downstream from site I11 of the tR1 terminator (HpaII frag- ment from the nut+ plasmid pLS1; see Fig. 1). This additional DNA increased the distance between the nut site and the end of the fragment and also increased the number of Rho-

B C

1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6

FIG. 3. N suppresses pausing by RNA polymerase at tRZ. Transcription reactions containing RNA polymerase and NusA were programmed with Hinfl DNA fragments from either pLS1 (nutR’; A and R ) or pLSl1 (nutR-; C). Reactions contained N and Rho factor, as indicated. Initiation by RNA polymerase was synchronized by the use of rifampicin (see “Experimental Procedures”), and transcription was allowed to proceed for the indicated times (in seconds). Transcripts were resolved on a urea-polyacrylamide gel and visualized by autoradiography. The positions of transcripts generated by RNA polymerase paused at various sites in tRI and of run-off transcripts are indicated.

Host Factors and Antitermination by X N Protein 19421

L

0 c 0 ; g 2 5 [r NusA NusA, NusB, I S10, NusG ' u u

S

[N] nM : I 9. 0 8 16 .40- 80 1601!.0 8 16 40 80 1 6 0 0 0 E

z z

RO - 7

- 586 - 527

- 404

lR1 [ - 371

335 - - 309

14- - 2 4 2 276 -

1 2 3 4 5 6 7 8 9 10 1 1 12 1314

FIG. 4. NusA is not sufficient for efficient antitermination by N at distal sites in tRZ. Transcription reactions containing E. coli RNA polymerase and programmed with the HpaII DNA fragment from pLS1 (nut+ template; see Fig. 1) contained either NusA alone (lanes I-7), or NusA, NusR, S10, and NusG (lanes8-14). All reactions (except lanes I and 14) contained Rho. The indicated concentrations of N were added to the reactions. The electrophoretic mobilities of labeled denatured DNA restriction fragments that were run on the same gel are indicated on the right of the panel (short lines, plain type). Also indicated on the right (long lines, bold type) are the positions of run-off transcripts produced and analyzed in a separate experiment and compared to the same DNA markers and Rho- terminated transcripts. The run-off transcripts used as RNA size standards were made on templates derived from pLSl DNA that was cut with HpaII and one other restriction enzyme that cuts within the tRI region. The restriction enzymes used and length (in nucleotides (nt)) of the run-off transcripts produced were: BstXI, 276; NdeI, 335; HinfI, 371; HpaII, 586. Transcripts terminated in the tR2 region and run-off transcripts are indicated to the left of the panel. The Rho- terminated transcripts similar in mobility to the 371 nt marker correspond to tR1 termination site IV (Lau and Roberts, 1985). Some transcripts terminated downstream of sites IV and V were also seen (indicated by the dashed line on the left). The arrow on the right of the figure indicates a NusG- and Rho-dependent termination site near nutR' (compare lanes 1, 2, and 8). Termination at this site is not affected by N.

dependent termination sites (Morgan et al., 1983; Chen and Richardson, 1987) downstream of the nut site. In the presence of only NusA and Rho, transcription was terminated a t sev- eral sites within the tR1 region (Fig. 4, lane 2). In reactions containing NusA, NusB, S10, NusG, and Rho, two smaller transcripts were detected in the vicinity of the nut site (Fig. 4, lane 8, indicated by an arrow). Therefore, the presence of one or more of the host factors, NusB, S10, or NusG, enhanced the termination activity of Rho. In experiments that will be published elsewhere, we have found that NusG does indeed interact directly with Rho factor and increase its termination activity.' In the absence of Rho, transcription did not termi- nate, and the amount of run-off transcription was the same regardless of whether the reaction contained only NusA or contained all four host elongation factors (Fig. 4, compare lanes 1 and 14).

As the concentration of N in the reaction was increased in the presence of NusA alone, the shorter terminated tran- scripts were converted to longer terminated transcripts, and a small amount of run-off length transcript was observed (Fig. 4, lanes 3-7). However, even at the highest concentration of N (lane 7,160 nM) most transcripts still terminated a t various sites within tR1. In contrast, as the concentration of N was increased in reactions containing all four elongation factors,

I d . Li, S. Mason and J. Greenblatt, manuscript in preparation.

termination in the tR1 region was efficiently suppressed, and the amount of run-off length transcript was substantially increased (Fig. 4, lanes 9-13). The distance from the nut site to the end of the DNA template used in Fig. 2 was 113 base pairs, whereas the distance from the nut site to the end of the DNA template used in Fig. 4 was 327 base pairs. Thus, although N and NusA sufficed for antitermination over a very short distance downstream from the nutR site (up to about 100 base pairs), the additional factors, NusB, S10, and NusG, greatly increased the efficiency of antitermination over larger distances.

Antitermination by N Is Highly Processive in the Presence of All Four Host Elongation Factors-Transcription from the pR and pL promoters must normally persist over great dis- tances on the X chromosome in vivo (Dambly and Couturier, 1971; Greenblatt, 1972). For example, transcripts must be elongated at least 7000 base pairs from the pR promoter to the end of gene Q. Since the elongation factors NusB, S10, and NusG had a large effect on antitermination by N over a relatively short distance, as demonstrated in Fig. 4, we wanted to assess the effects of these factors on transcription over the much larger distances that are characteristic of transcription i n vivo. Therefore, "P-labeled RNA produced in reactions containing X DNA as template was hybridized to various X DNA fragments that were immobilized on nylon membranes. The positions on the X chromosome of the DNA fragments used in these assays are shown in Figs. 5-8. The sizes of these DNA fragments and their distances from the pR and pL promoters are listed in Table I.

We initially examined the collective effect of the host factors NusB, S10, and NusG on the ability of N and NusA to prevent transcription termination (Fig. 5). Three fragments from the rightward operon and four fragments from the leftward operon were excised from X DNA, purified, and directly blotted onto nylon membranes. The distances of the various fragments from the pR andpL promoters ranged from

TABLE I X DNA fragments used in hybridization experiments

X DNA fragment Coordinates of Size Distance from end points" promoterh

bp pR operon'

R l a 37,937-38,214 277 0 R l b 38,103-38,214 111 80 R2 38,214-38,358 144 191 R3 39,994-40,598 604 1971 R4 40,810-41,117 307 2787 R5 41,117-42,251 1134 3094 R6 44,972-45,679 707 6949 R7 45,679-46,366 687 7656

L1 34,697-35,051 354 531 L2 33,498-34,319 821 1263 L3 32,964-33,585 621 1997 L4 31,702-32.407 705 3175 L5 28,052-29,040 988 6542

cl 37,584-37,937 353 "The locations of restriction enzyme sites on the standard X

chromosomal map (Daniels et al., 1983). The distance downstream from either the p R or p L promoter to

the promoter-proximal-most endpoint of each restriction fragment is given in base pairs.

Fragment R la was used only in Fig. 5. Fragments R l b and cl were used in Figs. 6-8, and not in Fig. 5. For unexplainable reasons, hybridization of RNA to Fragment R2 was poor and therefore Frag- ment R2 was omitted from all experiments.

,+The intensity of the hybridization signal of the cl fragment could be used to normalize different reactions within a single experiment.

pL operon

control fragmentd

19422 Host Factors and Antitel

"- L4 L3 L2 L1 Fii

" R3 Rb

1 / 1 1 Factors N ( n h \ \ a A -

I

b B

C

A 50

ABSG -

d ABSG 50 I

a A - I b A 500 "

C C ABSG - I

FIG. 5. The elongation factors NusA, NusB, S10, and NusG allow processive antitermination by N in the rightward and leftward operons of X. A , map of the early region of the X chro- mosome, indicating the positions of promoters (pli, p L ) , terminators ( ! H I , tH2. t L I , tL2), and DNA fragments used in this experiment. Vertical numbers under the X chromosome ( thick stippled line) are the standard A coordinates (Daniels et a/., 1983) and indicate the end points of the small X DNA fragments used for hybridization ( t h i n solid or stippled lines). Arrolvs indicate directions of transcription from the pl, and pH promoters. R and C , transcription reactions programmed with X DNA contained RNA polymerase, Rho factor, and either NusA alone or NusA, NusB, S10, and NusG. Reactions b and d also contained N, as indicated, a low concentration in R (50 nM) and a higher concent,ration in C (500 nM). Labeled RNA produced in these reactions was hybridized to the indicated X DNA fragments that had been immobilized on nylon membranes. Hybridized RNA was visualized by autoradiography.

100 to 2500 base pairs for the rightward operon and 500 to 3500 base pairs for the leftward operon (Fig. 5A; Table I). In transcription reactions containing NusA, but no NusB, S10, or NusG (Fig. 5B, lanes a and b) , N (50 nM) stimulated only a very small increase in the amount of RNA hybridizing to DNA fragments from either operon. On the other hand, in reactions containing NusA, NusB, S10, and NusG (Fig. 5B, lanes c and d ) , N stimulated a large increase in the amount of RNA that hybridized to all DNA fragments located far from the promoters. This was true even for fragments R4 and L4, which are 2-3 kb pairs downstream from the p R and p L promoters. The only exception was the promoter-proximal fragment R1 of the pR operon, since this fragment lies just downstream of pR and upstream of the nutR site. Transcrip- tion of this region should not be stimulated by N. Therefore, the presence of NusB, S10, and NusG in transcription reac- tions greatly increased the distance over which antitermina- tion by N could occur.

Recently, Sullivan and Gottesman (1992) suggested that NusG was not required for antitermination by N a t Rho- independent terminators i n vivo. In their experiments, N was expressed from a pLac promoter, and the concentration of N i n vivo was likely to have been in the range of 1-10 p M (Greenblatt et al., 1980). In our experiments, when the con- centration of N in the reactions was increased from 50 to 500 nM (Fig. 5C), NusA was sufficient to allow antitermination through the region that was tested, especially for the p R operon (Fig. 5C, lane b ) , and the requirement for the elonga-

-mination by X N Protein

tion factors NusB, S10, and NusG was decreased (Fig. 5C, compare lanes b and d ) . This was true both for Rho-dependent (tR1 and tL1) and Rho-independent ( tR2) terminators in either operon. Therefore, a 500 nM concentration of N may be sufficiently high so that it bypasses the requirement for certain host factors in our in vitro reactions. Furthermore, at more physiological concentrations of N that are sufficient for function i n vivo (50 nM in Fig. 5B; Greenblatt et al., 1980), all four elongation factors, NusA, NusB, S10, and NusG, were absolutely required for long distance antitermination.

In order to ensure that the transcription, we were measuring was originating from the p R and p L promoters, transcription reactions were assembled in the presence of the X CI repressor protein. The CI repressor binds to the OL and OR operator sites of X and specifically inhibits transcription from the p L andpR promoters (Steinberg and Ptashne, 1971). This exper- iment was important since double-stranded DNA fragments were used for hybridization probes in all of our experiments. In this and subsequent experiments we used some additional DNA fragments located as far as 7000 bp downstream from the p L and p R promoters (see Table I and Fig. 6A). Each fragment was cloned into the plasmid pTZ19R to facilitate quantitation and the same molar amount (400 pmol) of each uncut plasmid DNA containing a X DNA insert was denatured and blotted onto nylon filters. The results are shown in Fig. 6B. Once again, N greatly stimulated the transcription of all nut site-distal DNA fragments in reactions containing NusA, NusB, S10, and NusG (compare lanes a and b) . Stimulation by N was not due to an increase in the amount of transcription initiation since transcription of the c l gene and transcription of fragment R1, located immediately downstream from pR, were not stimulated by N. When the CI repressor protein was added to a reaction containing N and all four host factors at a concentration that was sufficient to repress transcription from the pR and p L promoters, the amount of RNA that hybridized to all of the fragments in both operons was drast- ically decreased (compare lanes a and c ) . Transcription of the c l gene itself was repressed slightly by the CI repressor, presumably because of the high concentration of CI protein we used in this experiment (7.8 p ~ ) . These results indicated that most of the transcription we were measuring in these reactions originated from the p L and p R promoters. There- fore, the stimulation of transcription by N in reactions con- taining all four host factors resulted from increased length of transcripts originating at the p L and p R promoters and not from transcription initiation elsewhere on the X DNA tem- plate.

Transcription in the presence of N and all four host factors was quantified by densitometry and compared with that in the absence of N. The results of three separate experiments are shown in the graphs of Fig. 6C. These results indicated that N had a 2-10-fold stimulatory effect on transcription through various regions of the leftward and rightward operons and that this effect decreased a t greater distances from the promoters. This decrease in antitermination at greater dis- tances has also been observed in vivo (Gottesman et al., 1980). The amounts of RNA that hybridized to fragments R6 and R7, located 6-7 kb pairs downstream from the pR promoter, were less affected by the presence of N and CI protein (see Fig. 6, B and C), probably because these fragments are down- stream of the late operon promoter pR', which is not affected by CI or N, and would also hybridize to transcripts originating from that promoter.

NusA, NusB, SlO, and NusG Are All Required for Processive Antitermination by N-The effect of each host factor on processive antitermination was assessed by assembling tran-

Host Factors and Antitermination by X N Protein 19423

A

B

C

nutL nutR p?- - -

-e

tL3 tL2 tL1 1 pL pR 1 tR1 tR2 tR3tR4 tR' . b b

- L5

", L4 L3 L2 L1

-1

cl R1 -"

R3 R4 R5 . . ,. . .,. ....... ...I ....

R6 R7

a

b

C

-N,-cl '

!

i

.- L m 10

(r a, -

- N L5 L4 L3 L2 L1 R1 R3 R4 R5 R6 R7

pL Fragment Number pR Fragment Number FIG. 6. Transcript length is increased in the presence of NusA, NusB, S10, and NusG. A , map of the X chromosome showing the

positions of promoters, terminators, and X DNA fragments used for hybridization in this experiment. R, reactions were programmed with X DNA and contained RNA polymerase, Rho factor, NusA, NusB, S10, and NusG. Some reactions contained the X N and cI repressor (7.8 pM) proteins, as indicated. Labeled transcripts produced in the reactions were hybridized to plasmids containing the indicated X DNA inserts. Hybridized RNA was visualized by autoradiography. C, quantitation of hybridized RNA based on densitometric scans of multiple film exposures. Three separate experiments were quantified. Each hybridization signal was normalized to the intensity of the signal for the cI control fragment in each reaction and to the number of thymidine residues in the coding strand of each DNA fragment. For comparison, the three experiments were standardized to the intensities of the signals for p R operon fragment R1 (separately for + and - N reactions). The experiment shown in R was not quantitated, although the overall pattern of hybridization intensities was the same. Solid circles indicate reactions containing N. Open circles indicate reactions lacking N.

scription reactions from which single proteins were individ- ually omitted. The labeled RNA produced in these reactions was again hybridized to plasmids containing X DNA inserts (Fig. 7A) . Once again, RNA made in the presence of N and all of the host elongation factors (Fig. 7B, lane a ) produced more intense hybridization signals on all DNA fragments, except the c l gene fragment and the p R promoter proximal fragment R1, when compared with the hybridization signals produced by RNA from the reaction lacking the N protein (Fig. 7B, lane b). The omission of NusA, NusB, or S10 from the reaction (Fig. 7B, lanes c-e) was similar in effect to the omission of N and caused a drastic decrease in the amount and length of RNA produced in the reaction. On the other hand, when NusG was omitted from the reaction (Fig. 7B, lane f ) , there was little reduction in the amounts of RNA hybridizing to fragments L1 and L2 in the leftward operon (also see Fig. 70) . This reflected the ability of NusG to

enhance termination by Rho a t sites located upstream of the L1 and L2 fragments.' However, NusG was still required for N to stimulate transcription of the more distal fragments, R3-7 and L3-5, in each operon. The amount of labeled RNA hybridizing to each fragment from each reaction was quanti- fied and normalized to both the hybridization signal for the c l gene control fragment and the number of thymidine resi- dues in the coding strand of each fragment (see Fig. 70 ) . These results indicated that all four host elongation factors, NusA, NusB, S10, and NusG, are required for processive antitermination by the X N protein in both the rightward and leftward early operons of X.

All Four Host Factors Are Required for Processive Antiter- mination ut Rho-independent Terminators-We have shown that NusA, NusB, S10, and NusG are necessary and sufficient for processive antitermination by N on X DNA in the presence of Rho. To assess the requirement for each of these factors in

19424 Host Factors and Antitermination by X N Protein

FIG. 7. NusA, NusB, S10, and NusG are all required for processive antitermination. A, map of X DNA fragments used for hybridization. R and C, transcription reactions were pro- grammed with X DNA. The complete reaction ( a ) contained RNA polymerase, N, Rho, NusA, NusR, S10, and NusG. In each of the other reactions factors were omitted, as indicated. Labeled tran- scripts were hybridized to immobilized plasmid DNAs containing the indicated X DNA inserts and visualized by auto- radiography. D, quantitation of the hy- bridization signals from the reactions a- f in R, showing the effect of each factor on antitermination. 0, complete reac- tion; 0, -N; X, -Nus& W, -NusG; A, -NusB; 0, -SlO.

b > + I 1

C B

d . .

e

f

a

b C

D

- N I -NusA I '

-Nus9 1 1 ' 1 #

I I -s10 1 I f l -NusG

I . .

-14

-12

-10

8

6

4

2

-Rho

-N, -Rho

40 \ _i 20 12

lo/ IQ O J

L5 L4 L3 L2

pL Fragments

antitermination a t promoter-distal Rho-independent termi- nators, we used the same assay to assess transcription in reactions lacking Rho factor. When Rho was omitted from reactions, N still had a striking effect on transcription of fragments R4-7 of the pR operon (Fig. 7C, lanes a and b) . This is probably due to the presence of a strong Rho-inde- pendent terminator, tR2 (Das and Wolska, 1984; Schmidt and Chamberlin, 1987), in the region between fragments R3 and R4. On the other hand, N had little effect on transcription of the pL operon in the absence of Rho (Fig. 7C, lanes a and b ) , possibly because of the lack of strong Rho-independent ter- minators in this region of the pL operon. The tL2 terminator, which was shown to be a Rho-independent terminator in vivo (Das et al., 1983), only exerted a weak Rho-independent effect in our in vitro assay (Fig. 7C, compare fragments L2 and L3 in lanes a and b) .

To determine which host factors were required for antiter-

L1 dl d3 d4 d5 d6 d7

pR Fragments

mination by N a t tR2, transcription reactions were assembled from which single factors were individually omitted in the absence of Rho. The transcripts produced in these reactions were hybridized to X DNA fragments derived from the right- ward operon (Fig. 8). Again, transcription in the absence of N abruptly stalled beyond fragment R3, presumably a t tR2 (compare Fig. 8B, lanes a and b) . The omission from the reaction of NusA (Fig. 8B, lane c ) , however, allowed some transcription to proceed through the tR2 region to fragment R5. Greater read-through of tR2 in the absence of NusA was expected because termination at tR2 is stimulated by NusA (Schmidt and Chamberlin, 1987). A quite similar pattern of transcript hybridization was observed in the reaction contain- ing only RNA polymerase (Fig. 8B, lane h). In the absence of NusA, transcription terminated between fragments R5 and R6, probably a t tR', a strong Rho-independent terminator located between fragments R5 and R6. The addition of NusA

Host Factors and Antiter

PR' r---*

\ \ \ \ // a complete

b -N

C -A

d -B

B e -S

f - G

g -A,+Al " ,..

h R alone

I t

FIG. 8. NusA, NusB, S10, and NusG are all required for processive antitermination by N at Rho-independent termi- nators. A, map showing X DNA and fragments used for hybridization. H, reactions were programmed with X DNA. The complete reaction ( a ) contained RNA polymerase, N, NusA, NusB, S10, and NusG. In each of the other reactions single factors were omitted, as indicated. In reaction g the NusAl mutant protein was substituted for wild type NusA. Reaction h only contained RNA polymerase. Labeled tran- scripts were hybridized to plasmids containing the indicated X DNA fragments from the rightward operon and visualized by autoradiog- raphy.

purified from the nusAl (Friedman and Baron, 1974) mutant strain of E. coli (Fig. 8B, lane g), which does not allow antitermination by N, restored some of the termination at tR2. Unlike wild type NusA, however, the NusAl protein did not permit N to stimulate transcription of the distal fragments of the pR operon (Fig. 8B, compare lunes a and g).

The effect on transcription of omitting SI0 or NusG (Fig. 8B, lanes e and f) was similar to the effect of omitting N. Although the omission of NusB (Fig. 8B, lane d ) seemed superficially similar to the omission of NusA (Fig. 8B, lune c; see above), quantitation of the hybridization signals in Fig. 8 (data not shown) revealed that the omission of NusB was similar in effect to the omission of N (Fig. 8B, lane b ) . Therefore, these results indicated that NusA, NusB, S10, and NusG were all important for antitermination by N a t Rho- independent terminators located far downstream from the pR promoter in the rightward operon of X. This conclusion was particularly evident if one simply examined the hybridization signals for the most promoter-distal fragments, R6 and R7, in Fig. 8B.

DISCUSSION

Antitermination by the bacteriophage X N protein in uiuo involves a t least four host E. coli proteins: NusA, NusB, S10, and NusG (reviewed by Friedman, 1988; Roberts, 1988). How- ever, Whalen et al. (1988) showed that NusA was sufficient

-mination by X N Protein 19425

for N to suppress termination in vitro at the Rho-independent X terminator tR' when tR' was cloned immediately down- stream from the X nutL site. We have shown here that NusA is also sufficient for N to prevent termination in vitro at the Rho-dependent termination sites located less than 100 base pairs downstream from nutR in the X terminator tR1. In the absence of Rho factor the termination sites of tR1 are pause sites for RNA polymerase, and we have also shown that N and NusA can suppress pausing at these sites. The Q gene antitermination protein of X also suppresses pausing by RNA polymerase, both at the Q utilization site (Grayhack et al., 1985) and at the pause sites of tR1 (Yang and Roberts, 1989). I t seems likely that suppression of pausing by the X N and Q proteins is a critical aspect of their mechanisms of antiter- mination.

When we examined transcription through Rho-dependent termination sites of tR1 located more than 100 base pairs downstream from nutR, we found that N and NusA were not sufficient for antitermination a t these more promoter distal sites. The addition to the reaction of NusB, S10, and NusG, however, allowed efficient antitermination through the entire tR1 region. Moreover, we also showed that NusA, NusB, S10, and NusG were necessary and sufficient for processive anti- termination by N that persisted for very long distances of up to 7 kb in both the p L and pR operons.

Two strong Rho-independent terminators, tR2 and tR', are present in the rightward operon. The nin region of the pR operon (Court and Sato, 1969) also contains at least two other terminators, tR3 and tR4, in addition to tR2, both of which are believed to be Rho-dependent (Leason and Friedman, 1988; Cheng et al., 1991): In reactions containing all four elongation factors, N helps RNA polymerase to elongate through this entire region either in the presence or absence of Rho. Therefore, we have shown that the four host factors are necessary and sufficient for N to suppress termination in vitro at several Rho-dependent and Rho-independent termi- nators scattered over several thousand base pairs in the rightward operon of X. This processive antitermination re- flects the situation in uiuo, where N-modified transcription complexes originating from pR must transcribe at least through gene Q and can even transcribe the entire late gene region of X (Dambly and Couturier, 1971). There is, however, a distinct loss of processivity that is observed in vitro (see, for example, Fig. 6) and in vivo (Dambly and Couturier, 1971; Gottesman et al., 1980) over very long distances. I t is, perhaps, for this reason, as well as for proper temporal expression, that efficient transcription of the X late genes requires antitermi- nation by the X Q protein of transcription originating at the late promoter pR'.

Our observation that NusG is important for antitermina- tion in uitro at promoter-distal Rho-independent terminators (Fig. 8) seems contradictory to the observation by Sullivan and Gottesman (1992) that NusG is not important for anti- termination at Rho-independent terminators in uiuo. Several possibilities exist to explain this difference. First, it is possi- ble, as Sullivan and Gottesman (1992) suggest, that different host factors are required for antitermination at different terminators. In our experiments, we found that NusG was required for antitermination at all terminators that we tested in both X operons. However, the terminator used in the Sullivan and Gottesman (1992) experiments, X tl (Oppenheim et al., 1982), was not tested in our experiments. Another possibility is that an alternative host factor can substitute for NusG to support antitermination by N in uiuo. A third pos- sible explanation is that the production of N in uiuo at high

S.-W. Cheng and D. Friedman, personal communication;

19426 Host Factors and Antitermination by h N Protein

temperature by Sullivan and Gottesman (1992) using a pLac promoter may have bypassed the requirement for certain host factors. Indeed, we found that a high concentration of N in vitro could partly bypass the requirement for NusB, S10, and NusG. Moreover, as found by Peltz et al. (1985), temperature :an affect the requirements in uiuo for antitermination by N. They found that the boxA element of the nut site was required for antitermination by N at 42 "C, but not at 30 "C. In Eontrast, Zuber et al. (1987) found that they could delete most Df the important boxA sequence in a X nut site and still get sntitermination by N in uiuo. Their assays were also per- Formed at high temperatures, but their nut site deletion con- structs had different end points from those of Peltz et al. (1985). Therefore, we think that NusG is important for anti- termination by N at Rho-dependent and -independent ter- minators, but that conditions can be found where the require- ment for NusG, and perhaps also for NusB and S10, can be bypassed (Figs. 2, 3, and 5C).

Why is there a dramatic increase in the processivity of antitermination when NusB, S10, and NusG are added to reactions containing N and NusA? The simplest explanation is that all four host elongation factors are necessary for prolonged stability of the N-modified transcription complex. We have shown elsewhere that NusA, NusB, S10, and NusG 3re all important for the stable association of N and these Factors with RNA polymerase and the nut site RNA in N- modified transcription complexes (Mason and Greenblatt, 1991; Nodwell and Greenblatt, 1991). I t seems likely that the two critical sequence elements of the nut site RNA, boxA and box& bind different combinations of these factors (Ward et zl., 1983; Horwitz et al., 1987; Lazinski et al., 1989; Friedman ?t al., 1990; Nodwell and Greenblatt, 1991)* and that both 5oxA and b o d are important for processive antitermination (Horwitz et al., 1987; Li et al., 1992). We suggest that the werall stability of the N-modified form of RNA polymerase is a consequence of a large number of weak protein-protein snd protein-RNA interactions, and this stability is important for processive N-mediated antitermination (Mason and Greenblatt, 1991; Nodwell and Greenblatt, 1991).

Acknowledgments-We thank all the members of the laboratory for discussion and advice. We thank V. Nowotny for the gift of purified S10 and A. Astromoff and M. Ptashne for the gift of purified :I repressor protein. We also thank H. Krause, B. Andrews, J. Nodwell, and L. Desbarats for reading and commenting on this nanuscript.

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