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The Nut Site of Bacteriophage K Is Made of R.NA and Is Bound by Transcripuon Anutermination Factors on the Surface of RNA Polymerase

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The Nut Site of Bacteriophage K Is Made of R.NA and Is Bound by Transcripuon Anutermination Factors on the Surface of RNA Polymerase Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press The nut site of bacteriophage k is made of R.NA and is bound by transcripuon anutermination factors on the surface of RNA polymerase Justin Rea Nodwell and Jack Greenblatt Banting and Best Department of Medical Research and Department of Medical Genetics, University of Toronto, Toronto, Canada M5G 1L6 The boxA and boxB components of the k nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, Sl0, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA. [Key Words: Transcription antitermination; RNA footprinting, bacteriophage h] Received June 14, 1991; revised version accepted September 11, 1991. Shortly after bacteriophage h infects a cell or is induced 1986) and are known to be important for transcription from the prophage state, Escherichia coli RNA polymer- antitermination by N (Zuber et al. 1987). boxB is located ase initiates transcription from the h promoters PL and 9 or 10 bp downstream from the core boxA sequence. It p~, reads through the early genes N and cro, and is is a 15-bp sequence that has hyphenated dyad symmetry stopped by transcription terminators that are located and, hence, the capacity to form a stem-loop structure in downstream from the early genes. Once the N protein is single-stranded nucleic acid (Rosenberg et al. 1978). made, it modifies RNA polymerase molecules that are Four Escherichia coli proteins function as cofactors in transcribing the early genes so that they become resis- N-mediated antitermination: NusA, NusB, S10, and tant to transcription termination and read through the NusG (Friedman and Baron 1974; Keppel et al. 1974; terminators into the delayed early genes (Adhya et al. Friedman et al. 1976, 1981; Das and Wolska 1984; J. Li, 1974; Franklin 1974). The delayed early protein Q later R. Horwitz, S. McCracken, and J. Greenblatt, in prep.). causes a similar antitermination event during transcrip- N-modified transcription complexes assembled in vitro tion from the late promoter PR', resulting in the efficient contain N and all four host cofactors (Batik et al. 1987; transcription of the late genes. Thus, the order of expres- Horwitz et al. 1987; Mason and Greenblatt 1991; J. Li, R. sion of the genes of h during lytie development is deter- Horwitz, S. McCracken, and J. Greenblatt, in prep.). mined by their positions along the h chromosome rela- Antitermination by N in vitro at terminators located a tive to promoters and terminators and by the action of short distance downstream from a nut site requires only the antitermination proteins N and Q. the host factor NusA and the boxB component of the nut The modification of RNA polymerase N requires nut site (Whelan et al. 1988; J. Li, R. Horwitz, S. McCracken, sites, which are located in transcribed, but untranslated, and J. Greenblatt, in prep.). Antitermination by N in regions between the promoters and first terminators of vitro that persists far downstream from the nut site and the early operons (Friedman et al. 1973; Salstrom and reflects N function more closely in vivo, requires boxA Szybalski 1978). The nutL and nutR sites of bacterio- and the additional host factors NusB, S10, and NusG in phage h have two genetically defined components: boxA addition to N, NusA, and boxB (J. Li, R. Horwitz, S. and boxB. The k boxA sequence is traditionally defined McCracken, and J. Greenblatt, in prep.). as CGCTCTT (Olson et al. 1982; Peltz et al. 1985); how- The results of several indirect experiments have im- ever the 9 or 10 nucleotides immediately downstream of plied that the functional form of the nut site may be in it (the extended boxA homology region) are evolution- the nascent transcript RNA rather than the DNA of the arily conserved in lambdoid bacteriophages (Morgan chromosome of the phage. Ribosomes translating across Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Nodwell and Greenblatt the nut site RNA in vivo inhibit antitermination by N (Olson et al. 1982; Warren and Das 1984; Zuber et al. 1987). Also, treatment of high-performance liquid chro- matograpy {HPLC)-purified N-modified transcription complexes with a large amount of ribonuclease T1 causes the loss of N and some of the NusA from the complex, indicating that the presence of these proteins in the complex is stabilized by RNA (Horwitz et al. 1987). To explain these results, we have proposed that N and one or more of the host antitermination proteins bind to the nut site in the nascent transcript and form a ribonucleoprotein complex that associates with RNA polymerase and renders it resistant to transcription ter- mination (Greenblatt 1984; Horwitz et al. 1987). We have reconstituted antitermination by N in vitro using purified proteins (J. Li, R. Horwitz, S. McCracken, Figure 1. (A) The right early transcript of bacteriophage k, in- and J. Greenblatt, in prep.) and have now used this re- cluding the positions of the oligonucleotides JN-1 and JL-1, constituted system to perform footprinting experiments which were used for primer extension. (B,C) The results of on the nut site RNA in elongating transcription com- primer extension with JL-1 and JN-1, respectively, on the RNA plexes. Our results indicate that the nut site RNA in the produced following various times of chain elongation in the nascent transcript binds one or more of the transcription presence and absence of the N protein. NusA, NusB, $10, and antitermination factors and forms a stable ribonucle- NusG were present in all of the reactions at the concentrations oprotein complex that is carried along in association listed in Materials and methods for footprinting experiments. with RNA polymerase during chain elongation. Results The RNA was partially digested with the single-stranded Reasoning that nut site RNA-protein complexes might RNA-specific ribonuclease M1 or the double-stranded normally only form during transcription, we set up ribo- RNA-specific ribonuclease V1 or it was partially meth- nuclease and dimethylsulfate (DMS} footprinting on the ylated with DMS. Because the primer extension assay RNA in active transcription complexes using conditions only detects methylation by DMS of N1 of adenine and in which processive antitermination by N in vitro re- N3 of cytosine, DMS is a single-stranded RNA-specific quires NusA, NusB, S10, and NusG (J. Li, R. Horwitz, S. reagent in this context. McCracken, and J. Greenblatt, in prep.}. Reactions were The elongation factors alone, in the absence of ribonu- programmed with pLS-1 (Lau et al. 1985), a plasmid con- cleases or DMS had no effect on the RNA {Fig. 2A, cf. taining a pR-nutR--tR1 insert derived from bacteriophage lanes 4 and 5; Fig. 2B, cf. lanes 7 and 8). In Figure 2A, the k (see Fig. 1AI. Transcription was synchronized by mak- ribonuclease cleavage and DMS methylation sites were ing use of rifampicin (see Materials and methods}. At detected with the primer JL-1, which hybridizes 100 nu- various times after chain elongation was initiated, the cleotides downstream of nutR. The nut site RNA was RNA was analyzed by primer extension with the 32p. protected against ribonucleases M1 and V1 (92% and end-labeled oligonucleotide primers JN-1 and JL-1 (see 93% protection, respectively) when the antitermination Fig. 1A), which hybridize to the RNA distal to the nut factors were added to the reaction {Fig. 2A, cf. lanes 2 and site. In this way we found that essentially all of the tran- 3 with lanes 6 and 7). For both ribonucleases the pro- scription complexes had passed the nut site after <40 sec tected region included boxB, bonA, and all of the nucle- of chain elongation at 37~ (see Fig. 1B, C). In the foot- otides in between them. A prominent DMS methylation printing experiments ribonuclease or dimethylsulfate site between boxA and boxB was also protected in the (DMSI was added to the transcription reaction after 1 presence of the antitermination factors [Fig. 2A, cf. lanes rain of chain elongation. After an additional 1 rain of 9 and 10). Cleavage by ribonuclease V1 and methylation incubation at 37~ the RNA was extracted from the re- by DMS upstream and downstream of the nut site were action and the ribonuclease cleavages or DMS methyl- not affected by the presence of N and the host factors. ation sites were detected by primer extension with a 32p. Many ribonuclease M 1 bands downstream of the nut site end-labeled oligonucleotide and AMV reverse tran- were somewhat suppressed in the presence of N, NusA, scriptase. All of the effects of the factors on the reactivity NusB, $10, and NusG. Several pieces of evidence indi- of the nut site RNA with the ribonucleases or DMS were cate that the downstream suppression was the result of a densitometrically quantitated. slight inhibition of ribonuclease M1 activity and not the Figure 2 shows footprinting experiments on the RNA interactions between the downstream RNA and the pro- synthesized in reactions programmed with pLS-1 DNA. tein factors. First, the reactivity of the downstream RNA Transcription was performed either in the absence of with ribonuclease V1 and DMS was not affected by the added antitermination factors or in the presence of N, factors {Fig.
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