T 7 Early RNAs are Generated by Site - Specific Cleavages ( E . coli RNA polymerase / initiation / termination / RNase III / rho factor )

Transcription of 17 DNA by purified Escherichia coli RNA polymerase without added factors produces long RNA molecules that begin near the left end of T7 DNA and terminate at the end of the early region. An endonuclease has been isolated from uninfected E. coli that cleaves these long RNAs at five specific sites to generate RNA molecules essentially the same as the early T7 RNAs observed in vivo. This sizing factor, which may be RNase III, can act during or after RNA synthesis. Synthesis of early RNA chains has been shown to start at three strong initiators, spaced about 150-200 base-pairs apart near the left end of 17 DNA. Thus, the five cleavages by sizing factor generate the five early messenger RNAs of T7 plus three overlapping RNAs from the promoter region. RNA chains that are started at two of the strong initiators begin with A; those started at the third begin with G. A few minor initiators have also been observed, from which only short chains seem to be synthesized. Their locations in T7 DNA have not been mapped. Rho factor does not appear to be needed to generate any of these early 17 RNAs. When T7 infects Escherichia coli, all transcription of T7 DNA is from left to right, relative to the genetic map (1, 2). The host RNA polymerase transcribes only the left-most 20% of T7 DNA, the early region (3-5). Several early RNAs are produced (3), but a stop signal at the right end of the early region normally prevents transcription by host RNA polymerase from continuing into the late region of T7 DNA (5). A new RNA polymerase, specified by gene 1 of T7, transcribes the late region of T7 DNA (6, 7). In vitro, all transcription by E. coli RNA polymerase starts near the left end of T7 DNA (8) and proceeds to the end of the early region, where specific termination produces RNA molecules almost 20% the length of T7 DNA (9). To account for the series of smaller RNAs transcribed from this region in vivo, it has been suggested that posttranscriptional modification of this large RNA may occur in vivo (9) or that termination factors such as rho (10) might cause RNA chains to be released at specific points within the early region without preventing transcription from proceeding (8, 9). Several workers (8, 11, 12) have shown that purified rho causes termination of chain growth within the early region of T7 DNA or of the closely related T3 DNA. Using purified RNA polymerase, with or without rho, we were unable to reproduce the electrophoretic pattern given by T7 early RNAs in vivo. Therefore, we searched for and found another factor, present in uninfected E. coli, that causes the T7 RNAs synthesized by purified RNA polymerase to be essentially the same size as the early RNAs produced in vivo. This factor acts during or after RNA 1559 synthesis by cutting the RNA chain at specific points, and is probably identical to RNase III (13). MATERIALS AND METHODS Materials. Carboxymethyl-cellulose Cellex-CM and Agarose (Bio-Gel A-0.5 M, 200-400 mesh) were purchased from BioRad Laboratories, Whatman brand microgranular diethylaminoethyl-cellulose DE 52 was obtained from H. Reeve Angel & Co. [14C]ATP (510 Ci/mol), ['y-32P]ATP (14.2 Ci/mmol), and [y-32P]GTP (5.15 Ci/mmol) were purchased from New England Nuclear Corp. Preparation of RNA Polymerase and Rho. E. coli RNA polymerase holoenzyme was prepared as described (14) with minor modifications. Termination factor rho was isolated according to the procedure of Roberts (10), as modified by Darlix et al. (15). Preparation of DNA. DNA to be used as template was isolated by phenol extraction of phage preparations that had been purified by banding to equilibrium in CsCl. The extracted DNA was dialyzed extensively against sterile 10 mM Tris * HCl (pH 7.9)-i mM EDTA to remove phenol. In Vitro Synthesis of RNA. The standard reaction mixture for RNA synthesis contained in 0.1 ml: 20 mM Tris HCl (pH 7.9), 10 mM MgCl2, 50 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 100 gg/mi of bovine-serum albumin, 40 ,g/ml of DNA, 20 ,g/ml of RNA polymerase, and 0.2 mM each of UTP, CTP, GTP, and ATP, which included one or more of [14C]ATP (10 Ci/mol), [y-32P]ATP (250 Ci/mol), or [7-32P]GTP (250 Ci/mol). Reaction mixtures made at 00 were transferred to 370 at zero time, rifampicin (5 Mg/ml) was added at 5 min to prevent further initiation (16). Incorporation of label had essentially ceased by 20 min, at which time an equal volume of 50 mM EDTA containing 200 ,ug/ml of stripped E. coli tRNA was added, followed by two volumes of ice-cold ethanol. After standing for at least 1 hr at -15°, the precipitates 'that formed were collected by centrifugation and dried under reduced pressure. Electrophoretic Analysis of RNA. Samples of RNA synthesized in vitro, or in vivo, were dispersed in 25 ,A of buffer containing 1% sodium dodecyl sulfate, heated for 2 min in a boiling water bath, and subjected to electrophoresis on slab gels of polyacrylamide or polyacrylamide plus" agarose, as described elsewhere (17). RNase III Assay. The standard reaction mixture for assay of RNase III has been described by Robertson et al. (13). 1560 Biochemistry: Dunn and Studier