Nucleic Acids Research

Volume 4 Number 4 April 1977 Nucleic Acids Research

Transcription in vitro of bacteriophage lambda 4S RNA: studies on termination and rho protein

Bruce H. Howard, Benoit de Crombrugghe, and Martin Rosenberg

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014, USA

Received 21 December 1976

ABSTRACT When bacteriophage Xplal8 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl- transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of Ti oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the Xpgal8 DNA template, ' with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t ' site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t ' termination site. INTRODUCTION Control of transcription requires that termination as well as initiation occur at appropriate sites encoded by the DNA template. Efficient termination insures functional independence of adjacent operons (1). Termination sites, in addition, appear to serve as loci for modulation of transcription within operons (2-6). The N and cro operons of bacteriophage X contain the termination sites L and i, respectively; antitermination by the N gene product is required for expression of portions of N and cro operons distal to these sites (2,7). The tryptophan operon of E. coli contains a transcription termination site, called an attenuator, which precedes the first structural gene (8-10); it appears that the efficiency of termination at this site is modulated to provide an independent regulatory locus for trp operon expression (5,6). Evidence from in vivo and in vitro studies suggests that termination of transcription, like initiation, is in many instances regulated by additional protein factors (2,11-13). Of such factors, the E. coli protein rho has been the most extensively studied and its role most convincingly demonstrated (14-18). Rho is required for recognition in vitro by RNA polymerase of the

827 O Information Retrieval Limited I Falconberg Court London Wl V 5FG England Nucleic Acids Research t and t sites in bacteriophage X (2,11). Although termination occurs in vitro at the trp attenuator site independently of rho, rho mutants exhibit markedly increased readthrough in vivo (17). The details of the termination process, viz. pausing of RNA polymerase, release of RNA and RNA polymerase, efficiency of termination, and effects on reinitiation of transcription, will become better understood only when transcription of individual operons is studied. Therefore, we have focused our attention on in vitro synthesis from XpL8 DNA of a discrete 4S RNA designated "oop" (19). This RNA, which is transcribed from a point near the origin of X replication (20,21), provides an attractive system for several reasons. First, the nucleotide sequence of the 4S RNA as well as the sequence of Xpga18 for approximately 35 nucleotides beyond the 3' end of the transcript have been determined (22). Second, the similarities between 4S RNA and an RNA

"leader" sequence in the tryptophan operon suggest that in vivo the 4S may serve as a leader and its termination site as an attenuator (23,24). There is evidence from in vivo studies on the induction of X lysogens that read- through from the 4S might encode RNA for establishment of X repressor synthesis (25). Third, in transcription reactions in vitro 4S synthesis is stimulated greater than 5 fold by the action of rho (26). This increase in 4S synthesis is of interest, since it mimics the marked increase in 4S RNA which occurs in vivo during induction of X lysogens (21). EXPERIMENTAL PROCEDURE

Materials: Alpha-32P-labeled ribonucleoside triphosphates (100 - 150 Ci/ mmol) were obtained from NEN. Rifampicin and E. coli tRNA were purchased from P.L. Biochemicals. Pancreatic DNase (RNase-free) was from Worthington. Materials for polyacrylamide gels from Biorad were used directly. Xpgal8 DNA and separated strands from X c1857 DNA were prepared as described previously (27). Rho protein, purified by the method of Roberts (2), was greater than 90% pure as estimated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. RNA polymerase, purified according to Berg (28), was a gift from R. Musso (NCI). The restriction fragment Hae III 1190 (which contains the 4S gene) was kindly provided by R. DiLauro (NCI). RNA transcription: Reaction conditions are detailed in legends to the individual figures. RNA synthesis was stopped by addition of 0.4 ml. of a mixture containing: TrisHCl, pH 7.4 (0.1 M), MgOAc (3 mM), E. coli tRNA (90 ug/ml), and pancreatic DNase (25 ug/ml). After at least 15 minutes at 00 C., NaOAc, pH 5.2 was added (0.15 M final concentration) and the RNA extracted with an equal volume phenol. The samples were EtOH precipitated,

828 Nucleic Acids Research lyophilized and then processed as described below for either gel electrophoresis or hybridization. Gel electrophoresis: RNA was resuspended in 30 ul sample buffer: Tris borate, pH 8.5 (80 uM), EDTA (25 mM), SDS (0.62), and urea (8 M). Electro- phoresis was carried out in 6X polyacrylamide (acrylamide/bis 19:1) slab gels containing 8 M urea for 2.5 hrs. at 150 volts. RNAs were detected by radioautography and quantitated by either of two methods: a) scanning of radioautograms on a Joyce-Loebl Microdensitometer or b) counting of excised bands. Hybridization: To separate 4S and cro from other ?ipga8 encoded transcripts, RNA was hybridized in 4XSSC (SSC: 0.15 M NaCl, 0.015 Na citrate, pH 7.0) at 670 C. for 16 hrs. to Schleicher and Schuell B6 nitro-cellulose filters containing the Rae III 1190 restriction fragment (0.7 pg). Unhybri- dized RNA was digested with Tl RNase (0.5 units for 30 minutes at room temperature), after which RNase activity was removed by treatment for 40 minutes at 450 C. with a solution containing 0.15 M Na iodoacetate, 0.1 M NaCl, and 0.1 M NaOAc (pH 5.2). Carrier tRNA was added and the RNA eluted from the nitrocellulose filters by heating at 950 C. in 1.5 ml distilled H20 for 6 minutes. HaeIII 1190 DNA was removed by digestion with pancreatic DNase (50 ug/ml) for 5 minutes at room temperature, and DNase removed by phenol extraction, followed by EtOH precipitation. To remove cro transcript, the RNA was then hybridized in 2XSSC at 650 C. for 10 hrs. to 20 ug X 1 strand DNA. RNA-DNA hybrids were trapped on nitrocellulose filters, eluted and digested with DNase as before. After phenol extraction and EtOH precipitation, the RNA was taken up in distilled 1120 for mapping. Analysis of oligonucleotides: Digestion with Tl RNase and mapping of oligonucleotides in two dimensions by electrophoresis on cellogel at pH 3.5 and homocbromatography on thin layer plates of DEAE cellulose were performed by standard techniques as previously described (29,30). RESULTS Efficiency of termination at t' As seen in Fig. 1 (lanes 1 and 2) transcription in vitro by E. coli RNA polymerase from Xpz&18 DNA yields a discrete 4S RNA species, which is easily separated on polyacrylamide gels from 6S and larger "heterogeneous" RNAs. Transcription of this 4S RNA initiates at the p ' promoter and terminates at a site designated t' (see Fig. 2). Figure 3 is an example of a two-dimensional Tl fingerprint of 4S RNA purified by polyacrylamide gel electrophoresis. The terminal oligonucleotides are U6A(OH)2 and U6AU(OH)2.

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1 2 3 4 5 6

6S-

4S-

Fig. 1. Transcription reactions contained in 0.05 il.: Tris Rd, pH 7.9 (0.04 M), KC1 (0.06 M), MgAc (10 mM), dithiothreitol (0.1 nM), EDTA (2 mM), =32P ATP (10 Ci/ol), GTP, UTP, and CTP (0.2 aM each), and RNA polymerase (25 ug/ml); rho protein (4 ug/ml) and template DNA, either XpLql8 (30 ug/ml) or the Hae III 1190 fragment (2 ug/ll) were added where indicated. Lane 1: Xpgal8 DNA, minus rho; lane 2: Xpgal8 DNA plus rho; lane 3: Rae III 1190 DNA, minus rho; lane 4: Rae III 1190 DNA, plus rho; lane 5: XpSal8 DNA, minus rho; lane 6: )p,al8 DNA, plus glycerol (5% final concentration).

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Xpga/8 PL tL PL

QA J GAL cI CRO 4S SR h 9S u 6S PR PR Fig. 2. Schematic representation of DNA tmplates (not to scale). Arrowr designate origins and polarity of transcripts. The stippled area between the 1 and h strands indicates the approximate position of the Hae III 1190 restriction fragment.

The sequence of the DNA contiguous with and distal to the 3' end of the 4S RNA has been determined (22); written as the 1 strand it aligns with the 4S 3' terminal oligonucleotide U6A(OH)2 as follows: 31...AAAAAATAACCACTCTT.. .5'. . .WUWUTUA Accordingly, failure of RNA polymerase to terminate at i,' will be detected by appearance of the Ti oligonucleotide U6AUUGp(G), as well as other Ti oligonucleotides if synthesis continues well beyond the 3' end of the 4S. To examine the efficiency of termination at i', we purified by hybridization the RNAs transcribed from the k' promoter from those initiated at other X promoters. The kL'-initiated RNAs were then digested with Tl RNase and the oligonucleotide products analysed. Figure 4 is a Tl fingerprint of RNA transcribed from Wlpgal8 DNA and purified by hybridization to a restriction fragment (Hae III 1190) which contains the 4S gene, then to X 1 strand. The readthrough oligo U6AUUGp(G) is seen at the lower right. The fingerprint is much more complex than the map of the 4S, with many additional minor oligonucleotides. Most of these minor oligonucleotides are 1 strand-specific and appear to reflect read-through from the 4S RNA. This result suggests that when the termination signal ,' is not utilized, much larger RNAs are synthesized. Some of the minor oligonucleotides are not 1 strand specific and probably represent cro operon message detected due to low level concentration of our X 1 strand preparation with X h strand. We have examined h strand- specific oligonucleotides from the region between the cro and 4S genes to exclude the possibility that any of the oligonucleotide U6AUUGp(G) derives from h strand transcript (not shown). To quantitate the fraction of RNA polymerase molecules which do not terminate at i,', the read-through oligonucleotide U6AUUGp(G), the 3' terminal oligonucleotide U6AU(OH)2 and two internal 4S oligonculeotides were scraped from the thin layer DEAE homochromatography plate and counted. In

831 Nucleic Acids Research

:491kagi U6A(OH)2 .ir:

4 I :, U6AU( OH )2 I. -:

4 I a. 4 0 I-r;f 2 0 AAOUCAUtCCUGp(G) 0C-s 0 I: pH 3.5

Fig. 3. Autoradiograph of 4S RNA Ti oligonucleotides. Alpha32P-labeled RNAs transcribed in vitro from XpgalB DNA in the presence of rho were fractionated over a 4Z polyacrylamide gel containing 8M urea. The 4S band was electrophoretically eluted, EtOH precipitated, then digested with Ti RNase. Mapping was carried out by electrophoresis on Cellogel in 8M urea at pH 3.5 (first dimension) and ascending homochromatography on thin layer plates of DEAE cellulose (second dimension). U6A(OH)2 and U6AU(OH)2 are the terminal oligonucleotides.

832 Nucleic Acids Research

(JUGp lqw -~~~UAJO

tI a- aL3:

I i*A~~~~AA'CCAUCC'UJ(Gt(G3 U>sAUUGpfGl

0 0

0 pH 3.5

Fig. 4. Autoradiograph of Ti oligonucleotides from the 4S region synthesized in the absence of rho. Transcription was carried out in 0.1 ml. containing: Tris HR, pH 7.9 (0.04 M), KC1 (0.06 M), MgAc (10 mM), dithiothreitol (0.1 mM), EDTA (2 mM), ATP, GTP, alpha32P UTP (35 Ci/m ol), and CTP (0.2 mM each), Xpga18 DNA (30 ug/ml), RNA polymerase (25 ug/ml), and rifampicin (10 ug/ml). After 2 minutes at 370 C. the reaction was terminated, processed, and the RNA hybridized sequentially to the Hae III 1190 restriction enzyme fragment and to X t strand as described in Materials and Methods. Ti RNase digestion and two-dimensional mapping were carried out as in Fig. 3.

Table 1 the results of this analysis are presented: the oligonucleotide U6AUUGp(G) is found to be synthesized in 15% the molar quantity of the internal 4S oligonucleotide AACUCAUCCUGp and 18% of the internal oligonucleotide U2Gp, which occurs 3 times in the 4S sequence. The readthrough oligonucleotide is 27% of the terminal oligonucleotide U6AU(OH)2. In this experiment transcription was stopped after 2 minutes. In other experiments transcription was allowed to continue for 20 minutes: there is no signifi- cant increase in the ratio of the read-through oligonucleotide to internal oligonucleotides after the longer incubation time (not shown).

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Table I

Oligonucleotide Moles alpha 32P/mole oligonucleotide CPM Readthrough (calculated) AACUCAUCCUG(G) 3 6209 15% UUG(A) UUG (U) 1.3 6689 182 UUG(C) U6AU(OH)2 6 6568 21%* U6AUUG(G) 7 2114

Table I. Quantitation of Ti oligonucleotides. Alpha32P UTP-labeled oligonucleotides shown in Fig. 4 were scraped from the thin layer DEAE cellulose plate and counted by Cerenkov radiation. *In the calculation of readthrough based on the value for the terminal oligonucleotide U6A(OH)2, allowance has been made for the decrease in that oligonucleotide due to incomplete termination at t'.

Effect of rho on termination at i' To assess the effect of rho on the efficiency of termination at t ', alpha 32P labeled RNA synthesized in the presence of rho was processed and mapped as before. The Tl oligonucleotide pattern from this RNA is shown in Fig. 5: it is very similar to the fingerprint of 4S RNA isolated from polyacrylamide gels (see Fig. 3); in particular, the read-through oligonucleotide U6AUUGp(G) is not present, indicating that rho increases the efficiency of termination at L,' to 100%. Other than minor contaminating spots, one difference is evident: in Fig. 5 the terminal oligonucleotide U6AU(OH)2 is missing (we have found the ratio of the terminal oligonucleotides U6A(OH)2 and U6AU(OH)2 to be somewhat variable). Rho increases the number of 4S transcripts As mentioned in the introduction, rho stimulates 4S synthesis greater than 5 fold when Xpga18 DNA is transcribed in vitro (Fig. 1, col. 1 and 2). This stimulation in 4S synthesis can also be demonstrated when the HaeIII 1190 DNA restriction fragment is used as a template (Fig. 1, col. 3 and 4). The only strong promoter in the Hae 1190 fragment is ,', hence the mechanism for the stimulation of the 4S appears to be defined by the inter- action between RNA polymerase and the 4S cistron. Three lines of evidence indicate that the increase in 4S RNA synthesis does in fact reflect an increase in the number of initiations at the h I promoter: a) the 5-fold difference can be accounted for only in part by

834 Nucleic Acids Research

U6A(OH)2

:.. I ::.

I : S~~~

....

0L CD 0

0 0 AACUCAUCCUGp(G) S pH 365 - --

* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.:.:

Fig. 5. Autoradiograph of Ti oligonucleotides from the 4S region synthesized in the presence of rho. Reaction conditions were identical to those in Fig. 4, except that transcription was carried out in the presence of rho (4 ug/ml) and the incubation continued for 20 minutes.

835 Nucleic Acids Research

the suppression of read-through which occurs in the absence of rho; b) although not evident from Figs. 4 and 5 due to differing lengths of exposure, rho does increase the yields of the 4S internal oligonucleotides in approximate proportion to the increase in the 4S band observed on polyacrylamide gels; and c) if rifampicin is added to reactions so that transcription is limited to a single round, rho causes an increase of about 1.3 fold in 4S RNA, consistent with its effect on readthrough. Possible mechanisms for rho stimulation of 4S synthesis Glycerol has been reported to enhance transcription from several promoters (31). Since glycerol is present in our rho preparations, this possibility was ruled out (Fig. 1, col. 5 and 6). It is important to point out as well that the rho-mediated effect on 4S synthesis, like termination of lambda 12S and 9S RNAs, requires the RNA-dependent beta-gamma ATPase activity of rho (32). This ATPase requirement renders it unlikely that the. increase in 4S RNA synthesis is due to a minor contaminant in our rho preparation. The stimulation of the number of initiations at k' caused by rho could result from any of several mechanisms. Rho could directly increase the rate of initiation at ,' by increasing the affinity of RNA polymerase for the k' promoter. Rho could slow the depletion of RNA polymerase molecules available to initiate at k'. Alternatively, rho could facilitate the release of 4S RNA and/or RNA polymerase from the i ' site, and thereby act to maintain the 4S cistron "open" for further transcription. Rho does not appear to enhance the affinity of RNA polymerase for ,' in a manner analogous to cyclic AMP receptor protein (CRP). CRP has been shown to increase the rate of preinitiation complex formation at the lac and gal promoters (33,34). We find that the rate of preinitiation complex formation at p' is, in contrast, rapid and independent of rho. At the concentrations of RNA polymerase and Xpga18 DNA employed in these reactions, approximately 50% of RNA polymerase molecules initiate at k' within 10 seconds (Fig. 6). Potentially, rho could increase 4S synthesis by extending the duration over which RNA polymerase is able to initiate new transcripts. It has been reported that depletion of active RNA polymerase molecules occurs in in vitro transcription reactions with X DNA as template and at low salt (0.1 M KC1) (35,36). (Consistent with this, in our reactions incorporation of label reaches a plateau after about 20 minutes, and addition of fresh template DNA does not cause renewed RNA synthesis.) If rho slowed the overall rate at which polymerase is inactivated, of course, synthesis of X

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20 40 60 80 100 120 SECONDS

Fig. 6. Rate of preinitiation complex formation at £L'~ Reaction mixtures contained in 0.05 ml.: Tris RC1, pH 7.9 (0.04 M), KCI (0.1 M), dithiothreitol (0.05 mM), E:DTA (2 miM), ATP, GTP, alpha32P tlTP, (3 Ci/mmol), and CTP (0.2 miM each, 1XpgalB DNA (30 ug/mi) and rho (4 ug/mi) where indicated. At zero time R1tA polymerase (25 ug/mi) was added and preinitiation complex formation allowed to proceed at 370 C. At the time indicated on the abscissa, MgAc (10 mM final concentration) and rifampicin (10 ug/ml) final concentration) were added to start transcription and prevent further preinitiation complex formation. Incubation was then continued for 20 minutes to allow completion of all transcripts. RNA was processed, fractionated on 5% polyacrylamide gels containing 8M urea and the 4S band quantitated as described in Materials and Methods. Minusro E* * plus rhoO <

transcripts in addition to the 4S RNA should be stimulated. We find that rho causes only a small (less than 1.5 fold) increase in the synthesis of X~ 6S, 9S and 12S RNA.s (Fig. 2. and unpublished data); thus a major effect of rho on RNA polymerase depletion would seem unlikely. We compared the kinetics of synthesis of the 4S, 6S and 9S RNAs. These transcripts were quantitated by determining incorporation of alpha 3plabel into the appropriate bands on polyacrylamide gels. In the presence of rho, 4S synthesis and 9S synthesis reach a plateau within 2 minutes (in the absence of rho, 4S synthesis is also complete in less than 2 minutes). The early plateau of both 4S and 9S synthesis in the presence of rho further suggests that rho does not prevent the inactivation of RNA polynerase. The interpretation of thes results is complicated by the finding that 6S synthesis continues in the same reactions with or without rho at an almost linear rate for at least 20 minutes (not shown). The reason for the marked difference between 4S and 6S kinetics has not been defined, but is of interest and may reflect important differences between the respective promoters. The 6S promoter could be recognized by a different subclass of RNA polynerase molecules than are the 45 and 9S promoters. Alternatively, the affinity of the )k 6S promoter for RNA polynerase could be much higher than that of the

837 Nucleic Acids Research

x 4S and 9S promoters, allowing 6S transcription to continue as the concentration of active RNA polymerase falls. We examined the possibility that a functional block develops within the 4S gene after the first round(s) of transcription. If such a block occurs, it should be relieved by the action of rho, i.e. rho should act to maintain the 4S gene open for multiple rounds of 4S synthesis. An experiment was carried out in which at 2 and 5 minutes after the start of transcription, reactions were 'pulse-labeled' by the addition of fresh RNA polymerase and alpha 32P-labeled nucleoside triphosphate. Incorporation of label was allowed to continue for 30 seconds, after which rifampicin was added to preyent further initiations (see Table II). The amount of label incorporat- ed into 4S is proportional to the number of initiations over the 30 second intervals measured. Since fresh RNA polymerase is added, it should be in excess during most or all of the 30 second interval; hence, the amount of 4S transcription should reflect the availability of the 4S promoter. The data are expressed as "rounds of transcription", where one round is taken as the amount of 4S synthesis in reactions to which rifampicin has been added. During the 30 second labeling periods at 2 and 5 minutes in the absence of rho (minus rho, 180-210 seconds and 300-330 seconds) only 0.5 rounds of 4S synthesis are completed. Rho increases the number of rounds of 4S synthesis to 2.2 - 2.3 rounds for each of the intervals. The latter value is close to the maximum predicted given half-time for initiation of 10 seconds (Fig. 6). We interpret this experiment as being consistent with the capacity of rho to maintain the 4S gene open for multiple rounds of transcription. The completion of only 0.5 rounds in the absence of rho suggests that, after the first round, either entry to the h promoter or elongation of the 4S RNA is inhibited by 75-80%. Release of 4S RNA The 4S RNA is only 81 nucleotides in length (23). Accordingly, a pro- longed pause by RNA polymerase at L' to form a ternary "termination complex" might inhibit transcription of the 4S cistron by succeeding polymerase molecules. The ability of rho to maintain the 4S cistron in an open state would be easily explained if rho is required for release of the 4S RNA and/or RNA polymerase from the t ' site. Several experiments were performed to test for the release of 4S RNA from the Wpga18 DNA template in the absence of rho. First, reactions were filtered through nitrocellulose filters at varying times after initiation of synthesis. As expected, essentially all high molecular weight RNAs, repre- senting transcription complexes, were retained on the filters. In contrast,

838 Nucleic Acids Research

Table II

Scheme: RNA polymerase 2 or 5 min. 32P ATP 30 secondse,, i 20 min. 4 unlabeled NTPs 30 Rif 30 toc TO 37 C. RNA polymerase 37 C.

Rounds of 4S RNA synthesis/labeling interval Rho 120 - 150 seconds 300 - 330 seconds 0.6 0.6 + 2.2 2.3

Table II. Effect of rho on the availability of the PL' promoter. Reaction conditions were as described in Fig. 1., except that transcription was initiated in the absence of alpha32P-labeled nucleoside triphosphate; the template DNA was Xpgal8; rho (4 ug/ml) was present where indicated. At either 2 or 5 minutes alpha32p ATP (0.25 nmol; 110 Ci/mmol) and RNA polymerase (1.3 ug) were added. 30 seconds later rifampicin (10 ug/ml final concentration) was added to prevent further initiation. To determine rounds of 4S synthesis, standard control reactions were carried out in which alpha32P ATP label was present at the start of transcription. RNA was processed, fractionated on 5% polyacrylamide gels containing 8M urea and the 4S band quantitated as described in Materials and Methods. for all reaction times, 4S and 6S RNAs were recovered in the filtrates. Second, transcription reactions were passed over Sepharose 4S columns. X DNA, in association with high molecular weight RNA, was excluded by these columns. No 4S RNA migrated with the x DNA or in intermediate fractions; all 4S RNA moved more slowly than 6S RNA as predicted for the free species. Both the filter binding and gel filtration experiments were carried out at 40 C and 230 C and under high and low salt conditions. Third, transcription reactions were sedimented directly at 140,000g in air-driven ultracentrifuge (Airfuge ). Maximum g forces were reached 1 minute after initiation of transcription and maintained for 5 minutes; this was suffi- cient to pellet 30 percent of X DNA and associated high molecular weight RNAs. All 4S RNA remained in the supernatant fraction. These different experiments strongly suggest that 4S RNA is released from the Xpga18 DNA template independently of rho. We estimate that the half-time for release of the 4S is less than 3 minutes (assuming that the pelleting of 10% of the 4S RNA could be detected in the micro-ultracentrifuge experiment).

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'DISCUSSION The foregoing experiments have characterized transcriptional termination by E. coli RNA polymerase at a specific site, iL', in the X genome. We report that in vitro in the absence of additional protein factors, termination occurs at the i site with an efficiency of about 80%. 4S cistron transcripts which are not terminated at t,' appear to be elongated into relatively high molecular weight transcripts. Rho protein acts to increase the efficiency of termination to 100%. The extent of read-through which occurs in vivo may differ, of course, from the 18% value presented here. Indeed, if rho activity in vivo is approximated by our in vitro reactions, rho might be expected to cause 100% termination in the absence of anti-termination factors. The finding that RNA polymerase requires no additional factors to overcome the t ' signal is interesting, since it raises the possibility that anti-termination at this site in vivo could be mediated by antagonism of rho rather than by a direct action on RNA polymerase. We have investigated the mechanism of the rho-induced stimulation of 4S synthesis. We find that, in the absence of rho, an inhibition to 4S RNA synthesis develops after the initial round(s) of transcription. This inhibition is not due to the formation of a stable ternary termination complex at IL since 4S RNA is released independently of rho. The major component of the rho-mediated increase in 4S RNA synthesis, about 4 fold, appears to reside in the ability of rho to maintain the 4S gene open for multiple rounds of transcription. A minor component of the increase is due to the abolition by rho of read-through at i'. Although active RNA polymerase molecules become depleted from our reactions, the overall rate of the depletion does not appear to be markedly affected by rho. Termination of transcription probably consists of a number of discrete steps, which include: 1) pause or arrest of RNA polymerase at specific sites on the DNA template; 2) release of RNA; and 3) dissociation of RNA polymerase from the DNA. At independent termination sites, these three reactions occur without added factors. At rho-dependent termination sites, rho presumably activates one or more of these steps. In the case of termination at the 4S site, reactions 1 and 2 appear to occur without rho. This suggests that rho is acting at reaction 3, i.e. the dissociation of RNA polymerase. We feel that the simplest model consistent with our results is that rho acts to release RNA polymerase from ' together with the 4S RNA, whereas in the absence of rho RNA polymerase remains (at least transiently) at the termination site. Given the small size of the 4S gene, it is quite possible that 840 Nucleic Acids Research

persistence of RNA polymerase molecules at t ' could cause slowing of -L further 4S RNA synthesis. Thus rho may stimulate 4S transcription by accel- erating the release of RNA polymerase from the 4S termination site.

ACKNOWLEDGEMENTS

We wish to thank Drs. R. DiLauro, M. Gottesman, S. Adhya, and R. Musso

for helpful discussions, Ms. Cathy Brady for excellent technical assistance, and Alana Muto for typing the manuscript.

REFERENCES

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28 Berg, D., Barrett, K., and Chamberlin, M. (1971) Methods Enzymol. 21: 506-519. 29 Sanger, F., Brownlee, G.G., and Barrell, B.G. (1965) J. Mol. Biol. 13: 373-398. 30 Brownlee, G.G., and Sanger, G. (1969) Eur. J. Biochem. 11: 395- 399. 31 Nakanishi, S., Adhya, S., Gottesman, M., and Pastan, I. (1974) J. Biol. Chem. 249: 4050-4056. 32 Howard, B. and de Crombrugghe, B. (1976) J. Biol. Chem. 251: 2520-2524. 33 de Crombrugghe, B., Chen, B., Anderson, W., Gottesman, M., Perlman, R., and Pastan, I. (1971) J. Biol. Chem. 246: 7343-7348. 34 Nakanishi, S., Adhya, S., Gottesman, M., and Pastan, I. (1975) J. Biol. Chem. 250: 8202-8208. 35 Fuchs, E., Millette, R.L., Zillig, W., and Walter, G. (1967) Eur. J. Biochem. 3: 183-193. 36 Goldberg, A.R. (1970) Cold Spring Harbor Symp. Quant. Biol. 35: 157-161.

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