Effect of salt on the transcription of T 7 DNA by RNA polymerase from T 4 phage - infected

Transcription of T7 DNA by T4 core enzyme with host sigma is more sensitive to KCI than that by host core enzyme with host sigma. When salt is added after initiation of RNA chains has occurred, it is not inhibitory. Salt affects the binding of T4 enzyme to T7 DNA to the same degree as the binding of host enzyme. Active preinitiation complex formation is inhibited more by salt with the T4 enzyme and the inhibition is temperature-dependent. INTRODUCTION Transcription of T4 DNA by RNA polymerase from T4 phage-infected cells has been shown to be salt-sensitive'. Part of the salt-sensitivity of T4 holoenzyme has been shown to be due to the presence of a salt-promoted inhibitor of transcription ' . The inhibitor is removed along with the sigma subunit on phosphocellulose chromatography. T4 core enzyme resulting from the phosphocellulose chromatography is also salt sensitive ' . This paper deals with the site of inhibition of salt on the core enzyme. The site of inhibition has been studied by coupling the T4 core enzyme with host sigma so that preinitiation complex formation can be more easily measured. (Host sigma was used because of greater availability. The sigma subunit from T4-infected cells is similar after removal of the above described inhibitor.) When T7 DNA is used as a template with the T4 enzyme the reaction is much more sensitive to salt than when host core enzyme and host sigma are used. Results on the effects of salt on DNAenzyme binding, on active preinitiation complex formation, as well as on the overall reaction are presented here. MATERIALS ANDMETHODS Host core enzyme and host sigma were prepared as described previously . T4 core *This investigation was supported by the Energy Research and Development Administration under contract with the Union Carbide Corporation. 877 C Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research enzyme was prepared from phage T4 am_47~ X 42~-infected E. coli as previously described . T7 DNA was isolated from 17 phage according to the procedure of Thomas and Abelson. For ossay of RNA polymerase, the reaction mixtures (0.2 ml) contained [ C]ATP (Schwarz/Mann) (0.25 mM, specific activity, 3400 cpm/nmol), UTP, CTP, and GTP (P-L Biochemicals, Inc.) (0.25 mM each), Tris buffer (20 mM, pH 7.8) , MgCl j (10 mM), bovine serum albumin (Schwarz/Mann) (100 pg), 2-mercaptoethonol (10 mM), T7 DNA (10 u.g), and enzyme. Incubation was for 10 min at 37° and determination of radioactivity incorporated into RNA was carried out as previously described^. The assay of RNA polymerase binding to T7 DNA was essentially as described by Mueller . The reaction mixtures (0.2 ml) contained Tris buffer (20 mM, pH 7.8) , MgCU (10 mM), 2-mercaptoethanol (10 mM), bovine serum albumin (100 \ig), 17 DNA (lOjjg), and enzyme. Control reaction mixtures lacked T7 DNA. After incubation for 10 min at 37°C, 3.9 yg of poly (dA-dT) (Miles Laboratories), 50 nmoles of [ 1 4 C ] ATP (specific activity, 4000 cpm/nmole) and 50 nmoles of UTP were added. The reaction was continued at 37°C for another 10 min and determination of radioactivity Incorporated into acid-insoluble material was carried out as described previously . The reactivity of poly (dA-dT) as compared to the control meosured the amount of unbound enzyme. For assay of preinitiation complex formation, reoction mixtures contained the components described above for the binding reoction mixtures. After incubation for 15 min at a given temperature, 2 ug of rifampicin (Calbiochem), 50 nmoles of [ C]ATP (specific activity, 8000 cpm/nmole), and 50 nmoles each of UTP, CTP, and GTP were simultaneously added. The reaction was continued at 37°C for another 5 min, and determination of radioactivity incorporated into RNA was carried out as previously described^. RESULTS A N D DISCUSSION Figure 1 shows the effect of KCI concentration on the overall synthesis reactions of T4 enzyme and host enzyme with T7 DNA as a template. The overall reaction with T4 enzyme is stimulated by low concentrations of KCI (0.05 M and 0.1 M ) , but is inhibited by concentrations higher than 0.1 M . The reaction with host enzyme is stimulated by KCI concentrations up to 0.2 M and inhibited by 0.3 M . (Similar results with host enzyme have been reported previously^.)