Sites , and DNA Sequences Surrounding the 6 S RNA Template of Bacteriophage

Abstract

A major product of the transcription of bacteriophage lambda DNA in vitro is the 6S RNA. This article presents a detailed mapping of restriction endonuclease cleavage sites about the region of the 6S RNA template within the lambda genome. Restriction fragments defined by these sites have been used to localize the 6S RNA template within the physical and genetic maps of the lambda genome. Nucleotide sequence analysis of one of these fragments has largely confirmed the nucleotide sequence of the 6S RNA reported previously and has indicated the sequence of DNA that immediately follows the 6S RNA template. This article reports the nucleotide sequence following a known site of transcription termination by RNA polymerase of Escherichia coli. Genetic analysis of bacteriophage lambda has been underway for a number of years and has revealed control mechanisms of potentially broad biological significance (1). In addition, the properties of lambda DNA as a template for transcription in vitro have come under intensive investigation in recent years (1-5). One striking early finding concerning the template activity of lambda DNA was that purified Escherichia coli RNA polymerase, in the absence of rho termination factor, transcribes a discrete RNA species, the 6S RNA, from the lambda DNA template (2). The nucleotide sequence of the 6S RNA is now known (2). The transcription of this RNA continues to be of interest since its promoter initiates transcription more efficiently in vitro than any other promoter in lambda DNA (6) and termination of transcription occurs with precision, presumably determined by specific DNA sequences near the site of termination. We are currently engaged in investigation of the 6S RNA to determine the nucleotide sequences within the DNA that control the transcription of this RNA and to determine what role the 6S RNA might play in vivo during the course of lambda infection. In this report we present a detailed mapping of restriction enzyme cleavage sites within the bacteriophage chromosome near the template for the 6S RNA. We have used the DNA fragments produced by these various cleavages to precisely locate the 6S RNA template both on the physical map of lambda DNA and with respect to known genetic markers deleted from certain defective lambda prophages. We have also used these fragments to confirm portions of the sequence of the 6S RNA and to analyze the sequence of the DNA beyond the 3'-end of this RNA. MATERIALS AND METHODS Materials. 32P-Labeled, radioactive lambda DNA was prepared from Xpgal8S7 bacteriophage (7) grown on E. coli strain C600 in medium containing 2-5 mCi of inorganic [32P]_ phosphate per liter. Phage were banded in CsCl density gradients, and DNA was extracted from these phage as described (2). Nonradioactive E. coli DNA bearing deletion mutants of the lambda prophage was prepared by phenol extraction from E. coli strains numbers 5061, 1153, 1118, and 509 (8), which were generously provided by Dr. I. Herskowitz. E. coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) was prepared by the method of Lebowitz et al. (2). EcoRI and EcoRII restriction endonucleases were prepared by the method of Yoshimori (9). Restriction endonucleases from Hemophiluss influenza D (Hind) (10), H. influenza F (Hinf), H. aegyptius (Hae) (11), H. parainfluenza (HpaII) (12, 13), and H. haemolyticus (Hha) were prepared by the method of Smith and Wilcox (10). All other enzymes were obtained from commercial sources. Preparation and Fractionation of DNA Fragments. DNA fragments of Xpgal8S7 [32P]DNA produced by digestion with various restriction enzymes were fractionated by electrophoresis on slab gels of various percentages of polyacrylamide, as described (14). Fragment bands in the gels were visualized by autoradiography using Kodak RPR54 x-ray film. DNA fragments were eluted from excised gel segments by grinding and soaking the gel for several hours in 1.5mM NaCl/1.5 mM Na citrate (pH 7.0) at 4°. RNA -DNA Hybridizations. Procedures for the fixation of DNA to nitrocellulose filters and for the hybridization of RNA to filter-bound DNA are described in ref. 14. Preparation of RNA and RNA Sequence Analysis. Transcription of lambda DNA for the preparation of 32P-labeled 6S RNA is described by Lebowitz et al. (2). 6S RNA was purified from total RNA produced in lambda DNA transcription by electrophoresis at pH 8.3 on 4% polyacrylamide slab gels containing 8M urea. Transcription of Hinf-3 fragment (see Results and Fig. 1) was performed as described in ref. 14, except that the fragment was denatured by boiling in water for 5 min and cooled before being added to the reaction mix. a-32PLabeled nucleoside triphosphates (specific activity 50-150 Ci/mmol) used in all transcriptions were purchased from New England Nuclear Corp. 1817 * Present address: Institut de Biologie Molhculaire, Universit6 de Paris, Paris, France. Proc. Nat. Acad. Sci. USA 72 (1975) XyPUI a XP90l a by by

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@inproceedings{SklarSitesA, title={Sites , and DNA Sequences Surrounding the 6 S RNA Template of Bacteriophage}, author={Jeffrey L. Sklar and Sherman Weissman and Richard Roberts} }