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 initia- tion 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 con- tains a transcription termination site, called an attenuator, which precedes the first structural gene (8-10); it appears that the efficiency of termina- tion 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 tran- scription of individual operons is studied. Therefore, we have focused our attention on in vitro synthesis from XpL8 DNA of a discrete 4S RNA desig- nated "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 synthe- sis (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 electro- phoresis 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 tran- scripts, 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 precipi- tation, 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. 829 Nucleic Acids Research 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). 830 Nucleic Acids Research 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 hybridi- zation the RNAs transcribed from the k' promoter from those initiated at other X promoters.