Bacteriophage T7 late promoters: construction and in vitro transcription properties of deletion mutants

volume 10 Number 20 1982 N u c l e i c A c i d s Research Bacteriophage T7 late promoters: construction and in vitro transcription properties of deletion mutants Kenneth A.Chapman* and Robert D-Wells* Department of Biochemistry, University of Wisconsin, College of Agricultural and Life Sciences, Madison, WI 53706, and 'Department of Biochemistry, University of Alabama, Birmingham Schools of Medicine and Dentistry, University Station, Birmingham, AL 35294, USA Received 17 June 1982 INTRODUCTION T7 RNA polymerase is a monomeric protein (105,000 daltons) which is synthesized very early during the infection of E. coli by T7 (1). It is both considerably smaller and less complex than the bacterial RNA polymerase, but nevertheless is responsible for the expression of both the middle (class II) and late (class III) T7 genes (2). The late promoters have a structure very different from bacterial promoters, consisting of a highly conserved uninterrupted sequence extending from -17 to +2 bps relative to the initiation site. Since the expression of the class II and class III genes is regulated at the level of transcription (3), the late promoters also are divided into class II and class III. Of the 17 late promoters which are mapped and sequenced, 10 are class II, 5 are class III and 2 are near the ends of the genome (4,5,6,7,8). There are two structural differences between class II and class III promoters which probably account for the differences in their expression. Class II promoters are all located in the 14-40% region of the T7 genome while class III promoters are all located to the right of 40% (9). © IRL Press Limited, Oxford, England. 0305-1048/82/1020-6331S2.00/0 Class III promoters have the conserved 6331 ABSTRACT The construction of plasmids containing T7 class I promoters with deletion mutants was described. Restriction fragments, ending at the Hinf I site located at position -10 in the promoter from 14.8% of the T7 genome, were cloned into pBR322. This produced the deletion of either the left or the right part of the promoter. The in vitro transcription properties of these plasmids were determined. Control plasmids were obtained by cloning wild type class II and class III promoters into pBR322. These plasmids also were used to compare the in vitro transcription properties of the two classes of late promoters. Much of the leftward part of a T7 late promoter can be deleted without abolishing activity, but deletion of the right part eliminates promoter activity. Class II, class III, and the mutated promoters have characteristic responses to changes in ionic strength, exogenous glycerol, and temperature. Nucleic Acids Research sequence from -17 to to +2 and extend the region of conservation from -22 to +6. In order to more fully understand the essential features of the conserved region, a series of T7 late promoters with deletion mutations was constructed. Since these mutant promoters were on plasmids, the isolation of sufficient amounts of templates for a series of quantitative i^ vitro transcription experiments was facilitated. MATERIALS AND METHODS Enzymes Eco RI, Bam HI, and Alu I were generously provided by S. M. Stirdivant, W. Zacharias, and P. F. Lambert, respectively. E. Seising. Micrococcus luteus DNA polymerase (11) was prepared by TA DNA ligase was purchased from New England Biolabs. A partially purified T7 RNA polymerase was prepared from T7 infected E. coli MO cells. A cleared lysate of the infected cells was brought to 0.2M NaCl in P-buffer and run through a phosphocellulose column as described (12). The active fractions were pooled, diluted to 0.2M NaCl with P-buffer (described in 12), and run through the column a second time. produced a 160 fold purification of the polymerase. This procedure A 37% increase in speci- fic activity was observed when the pooled fractions were concentrated by dialysis (vs. P-buffer, 50% glycerol, 50mM NaCl) possibly due to the removal of excess salt. When assayed as described (12), the final pool contained 827 units/ml at a specific activity of 2560 units/mg. Two methods showed no detectable RNase contamination of the T7 TNA polyraerase. In an in vitro transcription experiment, using a 4 fold greater concentration of polymerase than normally used, the incorporation of [ H]-CMP into acid insoluble RNA showed a linear rate of increase which leveled off after about 1 hour and showed no decrease after 4 hours. Also, [ H]-labeled RNA was incubated with an 8 fold greater than normal concentration of polymerase in the transcription buffer at 37° for 1/2 hour; this produced no detectable loss of acid insoluble counts. This preparation also has proved to be stable for at least 6 months when stored at -20°. Cloning Methods DNA fragments for cloning were isolated from polyacrylamide gels (13). If the sticky ends of the fragment and vector were incompatible, they were filled in with Micrococcus luteus DNA polymerase before ligating (14). Ligation reactions (15) and transformations (16) were essentially as 6332 Hinf I was purified by the procedure of Greene et a_l. (10) from frozen Haemophilus influenzae R. cells (gift of U. Mueller). Nucleic Acids Research described. Several restriction sites in the tet region of pBR322 were used for the cloning of the fragments. Clones of the desired fragments were found by screening the amp tet colonies by toothpick assays (17), followed by microscale plasmid DNA preparations (18) and restriction mapping. Plasmid DNA samples were prepared as described (19). The E. coli strain MO (14) was used for purification of plasmid DNA and for the preparation of the T7 RNA polymerase. Construction of Promoters with Deletion Mutations The 122 bp Eco RI fragment, which contains a class II promoter (Fig. 1 ) , was cloned into the Eco RI site of pBR322 in both possible orientations. A 98 bp Hae III fragment, containing the class III promoter from 46.5% of the T7 genome (5), was isolated from T7 DNA and cloned into the Bam HI site of pBR322. In Vitro Transcription Unless otherwise stated, all in vitro transcription reactions were done at 37° in 50 mM Tris-HCl, pH 7.6, O.lmM dithiothreitol, lOmM MgCl,, 2.5mM spermidine, and ATP, GTP, UTP, and CTP (0.2mM each) (plus 20 tiCi/ml [3H]-CTP). 122 bp IO7bp 241 bp EcoRI Hint I EcoRI PRW35I Figure 1. DNAs used in this study. pRW351 was constructed by cloning a 116 bp Hpa II - Tag I fragment, which was shown by N. Panayotatos to contain the class II promoter from 14.8% of the T7 genome (4), into the Eco RI site of pVH51 (21). The thick line represents promoter sequences. 6333 Three DNA fragments, containing either a late promoter or a part of a late promoter, were obtained from pRW351 (Fig. 1). Deletions of either the left or the right part of the promoter were produced by cloning the 107 bp or the 241 bp fragment into pBR322. In these clones, the base-pairs missing from the promoter are replaced by the vector DNA adjacent to the clone (...truncated)