Bacteriophage T7 late promoters with point mutations: quantitative footprinting and in vivo expression (original) (raw)
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Nucleic Acids Research, 1987
This paper describes the construction of 18 cloned bacteriophage T7 late promoters with single point mutations. In vitro transcription experiments were used to characterize the properties of these promoters. Since the mutated promoters are cloned into identical backgrounds, differences seen in the transcription assays are directly attributable to the point mutations. All of the mutated promoters are less active than wildtype, but they can be divided into two types. Type A mutations map from-4 to +1 and reduce promoter activity when the template is linearized or when 60mM NaCl is added to the reaction buffer. Type B mutations map from-9 to-7 and reduce promoter activity under all conditions tested. At several sites all three possible point mutations are available. At these sites we observed hierarchies of base pair preference, as determined by promoter activity, that may indicate that T7 RNA polymerase interacts with groups in the major groove.
Proc. Natd Acad. Sci. USA Vol. 78, No. 8, pp. 4936-4940, 1981
Downstream placement of a strong transcrip-tional termination signal has made possible the cloning of bacte-riophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo. The efficiency of interaction between Escherichia coli RNA polymerase and transcriptional promoters of E. coli varies within a wide range when measured in vitro (1). For unregu-lated promoters, the rate ofcomplex formation in vitro reflects promoter strength in vivo (1, 2). However, despite the identification of more than 80 different promoter sequences and extensive study of promoter-RNA polymerase interactions (for survey, see refs. 3-5), the contribution of specific structural features to the functional activity of such sequences is not understood. Promoters from various bacterial and viral sources have been cloned in E. coli, and their signal strength in vivo has been studied by using expression from distal promoterless sequences encoding (3-galactosidase ((-Gal) or other proteins (6, 7) as an indicator of promoter activity. Attempts to clone small DNA fragments carrying the strong promoters of bacteriophage T5, which in vitro far exceed other promoters in the rate ofcomplex formation with RNA polymerase and the rate of initiation of RNA synthesis (1, 2), have been unsuccessful; however, fragments of T5 DNA containing both a strong promoter and a strong termination signal have been cloned (8). Subsequently, electron microscope analysis has shown that transcriptional regions of several E. coli plasmids are organized in well-defined units where termination signals appear to balance transcription initiated at promoters ofdifferent strengths (9). Together, these findings suggested that the cloning of strong promoter signals from phage T5 or other sources might require the downstream placement of comparably strong termination signals. We report here the construction and analysis of bacterial plasmid vectors that enable the cloning of promoters of high signal strength; such cloning is made possible by the positioning ofa transcriptional termination signal downstream from the site ofinsertion ofsuch promoters. The constructed plasmids, which allow estimation ofthe strength ofpromoter signals in vitro and in vivo, contain indicator genes in positions that also permit selection for termination signals. Using these vectors, we have isolated a library of T5 promoter sequences suitable for biochemical and physical investigations of promoter function and also potentially useful for achieving high-level transcription of heterologous genes introduced distal to the promoter signals. MATERIALS AND METHODS Restriction endonucleases, E. coli DNA polymerase, and bac-teriophage T4 DNA ligase were purchased from several commercial sources, and reactions were carried out as suggested by the supplier. EcoRI synthetic linker and adapter sequences were obtained from Collaborative Research (Waltham, MA). Phagefd DNA (replicative form) and plasmid pAD16/30 containing a 28-base-pair (bp) HindIII/BamHI adapter sequence were gifts from H. Schaller. lac repressor was a gift from A. Riggs. Isolation of bacteriophage T5 DNA (2), plasmid DNA (10), E. coli RNA polymerase (2), and termination factor rho (11) have been described previously. The binding of RNA polymerase to promoters and subsequent analysis of the complexes by nitrocellulose filter binding have been described (2). Identification and isolation of lac operator containing DNA fragments by repressor binding utilized the procedure of Riggs et al (12). The conversion of protruding 5' single-stranded DNA extensions to blunt ends and DNA lig-ation reactions have been described (13). Synthetic linker and adapter sequences were present in 3-to 10-fold excess relative to the various DNA fragments. Transformation ofE. coli strains C600r-m' (our laboratory collection), the M15 deletion-mutant DZ 291 (obtained from A. V. Fowler), and BMH71-18, an MiS derivative carrying the laCjq mutation (obtained from B. Muel-ler-Hill), was carried out as described (14). Selection oftransformants involved plating on LB plates containing chloramphenicol (Cm, 20 Ag/ml), ampicillin (Ap, 100 gg/ml), or varying amounts of tetracycline (Tc; 2-70 tug/ml). Selection for presence of the lac operator or production of the a fragment of (3-Gal was carried out on plates containing the antibiotic plus 5-bromo-4-chloro-3-indolyl (3-O-galactoside at 40 ug/ml (15). Induction of lac expression by isopropylthio-galactoside was as described (15). In vitro and in vivo RNA was prepared and analyzed as described previously (1) except that [a-32P]UTP and [32P]phosphate were used for labeling in vitro and in vivo, respectively. In vivo RNA was isolated from plas-mid-containing C600 cells after a 10-min labeling period. RESULTS Experimental Strategy. We have constructed a family of plasmids (Fig. 1) that carry two DNA segments that can be brought under the control of a single promoter and are separated by an endonuclease cleavage site suitable for the cloning Abbreviations: bp, base pair(s); Cm, chloramphenicol; Tc, tetracycline; Ap, ampicillin; (B-Gal, 3-galactosidase. 4936 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact.
Biochemistry, 1987
The interactions of T7 R N A polymerase with T7 late promoters were studied by using quantitative footprinting with methidiumpropyl-EDTA.Fe(I1) [ MPE-Fe(II)] as the D N A cleaving agent. Class I1 and class I11 T7 promoters have a highly conserved 23 base pair sequence from-17 to +6. Among class 111 promoters the-22 to-18 region is also highly cQnserved. For a class I1 promoter, T7 R N A polymerase protects the-17 to-4 region from MPE-Fe(I1) cleavage; when G T P is present, protection extends from-17 to +5 (noncoding strand). For a class I11 promoter, protection extends from-20 to-4 and in the presence of GTP from-20 to +5 (noncoding strand). The protected regions for the coding strands of both promoters were nearly identical with that seen for the noncoding strands. The binding constant for the class 111 promoter is (4 f 1.5) X lo7 M-' and in the presence of GTP increases to (10 f 1.7) X lo7 M-'. These binding constants are about 1000 and 200 times greater, respectively, than values reported previously [Ikeda, R. A., & Richardson, C. C. (1986) Proc. Nutl. Acad. Sci. U.S.A. 83, 3614-36181. The differences in binding constants are probably due to t R N A and high salt used in those earlier experiments. Both tRNA and high salt (>50 m M NaCl and > 10 m M MgC12) inhibit the binding of the polymerase to the promoter. Optimal binding conditions occur at 2-5 m M MgClz and 0-10 m M NaC1. Finally, the binding constant between T7 R N A polymerase and nonpromoter D N A sites was determined to be (2.1 f 1.1) X lo4 M-l. E a r l y in the infection of Escherichia coli by bacteriophage T7, a new phage-encoded RNA polymerase transcribes th'e middle and late T7 genes from the class I1 and class I11 promoters, respectively (McAllister et al., 198 1; Studier & Rosenberg, 1981). These promoters have several features that distinguish them from bacterial promoters. One such feature is the extensive homology between the promoters, as seen in Figure 1 (Dum & Studier, 1983). Most striking is the 23 base pair perfect match between the five class I11 promoters and the consensus sequence. For class I1 promoters the-17 to +6 region shows extensive homology with the consensus sequence. In the +3 to +6 region for class I1 promoters, 87% of the bases are purines but only 45% of the bases match the consensus sequence. In the-17 to +2 region of the class I1 promoters, there is greater than 90% homology to the consensus sequence. However, within the-22 to-18 region, class I1 promoters show no homology to the consensus sequence, whereas class I11 promoters show 84% homology to the consensus sequence. One of the goals of this paper is to characterize which bases in the-22 to +6 region are nec-Another difference between T7 and bacterial promoters is that T7 promoter sequences are unusually long and uninterrupted. The length probably ensures that T7 promoters do not occur randomly in the bacterial host genome. Yet eien including this requirement, T7 promoters still appear to be'too long. This notion is supported by a recent statistical analysis that concludes that T7 promoters are overspecified
Journal of Molecular Biology, 1990
The bacteriophage T3 and T7 RNA polymerases are closely related, yet are highly specific for their own promoter sequences. To understand the basis of this specificity, T7 promoter variants that contain substitutions of T3-specific base-pairs at one or more positions within the T7 promoter consensus sequence were synthesized and cloned. Template competition assays between variant and consensus promoters demonstrate that the primary determinants of promoter specificity are located in the region from-10 to-12, and that the base-pair at-11 is of particular importance. Changing this base-pair from G" C, which is normally present in T7 promoters, to C'G, which is found at this position in T3 promoters, prevented utilization by the T7 RNA polymerase and simultaneously enabled transcription from the variant T7 promoter by the T3 enzyme. Substitution of T7 base-pairs with T3 base-pairs at other positions where the two consensus sequences diverge affected the overall efficiency with which the variant promoter was utilized by the T7 RNA polymerase, but these changes were not sufficient to permit recognition by the T3 RNA polymerase. Switching the-11 base-pair in the T3 promoter consensus to the T7 base-pair prevented utilization by the T3 RNA polymerase, but did not allow the T3 variant promoter to be utilized by the T7 RNA polymerase. This probably reflects a greater specificity of the T7 RNA polymerase ~ for base-pairs at other positions where the promoter sequences differ, most notably at-15. The magnitude of the effects of base substitutions in the T7 promoter on promoter strength (-llC >>-10C >-12A) correlates with the affinity of the T7 polymerase for the promoter variants, suggesting that the discrimination of the phage RNA polymerases for their promoters is mediated primarily at the level of DNA binding, rather than at the level of initiation.
JOURNAL OF BACTERIOLOGY, Oct. p. 70-77, 1985
Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around-10 and-33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around-10 (TAT AAT) and-35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be usedfor the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems. Promoters of the Escherichia coli system start synthesis of functional RNAs with vastly different efficiencies. Little is known, however, about the rules by which functional parameters are implemented within a promoter sequence. Despite our knowledge of more than 150 promoter sequences (10) and a wealth of genetic and biochemical data (19), we are still unable to make reasonable predictions on functional properties of a promoter from structural information alone. Consensus sequences of E. coli promoters derived from sequence compilations, have elucidated some important general features. However, synthesis of consensus promoters (5) have resulted in signals which are, at most, average in function (U. Deuschle and M. Kammerer, personal communication). This is not surprising if one considers the complexity of the process programmed by a promoter sequence as well as the fact that in the derivation of consensus sequences there is usually no value describing functional parameters given to individual sequences. We approached this problem in a different way. By selecting for the most efficient unregulated promoters in the E. coli system, we expected to reveal sequences which would exhibit pertinent structural features most clearly. The selection principles utilized for the identification of efficient promoters were the determination of (i) the rate of complex formation between RNA polymerase and promoter in vitro, (ii) the relative efficiency of RNA synthesis in vitro under competitive conditions, and (iii) the relative promoter strength in vivo. The in vitro analysis of promoter-carrying DNA fragments has been described previously (6, 7). For the in vivo study of promoters we developed cloning systems which allow the stable integration of strong promoters as well as the precise determination of their in vivo function (9, 21; U. Deuschle, M.S. thesis, University of Heidelberg, 1984). Of about 60 promoters tested (including those of coliphage T7, fd, and X) some of the most efficient signals were found in the genome of coliphage T5. Here we describe the application of the * Corresponding author. t Present address: F. Hoffmann-La Roche & Co. A.G., ZFE CH 4002 Basel, Switzerland. pDS1 vector system (21; Fig. 1) for the selective cloning of strong promoters, the identification and structural analysis of 11 promoters of the phage T5 genome, and some of the functional properties of these promoters. As can be seen from the results of this and previous studies (9), several promoters described here appear especially useful for the efficient in vitro synthesis of defined RNA species, and as some of the promoters accept 7-mGpppA as the starting dinucleotide capped RNAs can be directly obtained in vitro. This transcription-coupled capping allows an efficient and selective expression of cloned DNA sequences in vitro which has been found to be especially useful in studying the translocation of proteins into or through membranes (11, 23). MATERIALS AND METHODS Enzymes and chemicals. Restriction enzymes, T4 DNA ligase, calf intestinal alkaline phosphatase, and RNase Ti were purchased from Bethesda Research Laboratories, Gaithersburg, Md.; New England Biolabs, Inc., Beverly, Mass.; or Boehringer Mannheim Biochemicals, Indianapolis, Ind.; and T4 DNA kinase was obtained from H. Schaller (University of Heidelberg). Reactions were carried out as recommended by the supplier. The isolation of bacteriophage T5 DNA and E. coli RNA polymerase has been described previously (7). XhoI synthetic linkers were obtained from Collaborative Research, Inc., (Waltham, Mass.) and were present in ligation assays in a 20-fold molar excess relative to that of the various DNA fragments. [-y-32P]ATP and [a-32P] UTP were from Amersham & Buchler (Braunschweig, Federal Republic of Germany) and 7-mGpppA was obtained from P-L Biochemicals, Milwaukee, Wis. Plasmids and their nomenclature. The basic pDS1 vector system has been described previously, and here we follow previously proposed nomenclature (21). The identity of the promoters and terminators which have been integrated can be derived from the designation of the plasmid: pDS1/ PH207,tol describes a plasmid-carrying promoter PH207 in front of the coding sequence (dhfr) for dihydrofolate reductase (DHFR) and terminator to from phage lambda at site 1 (Fig. 1). Another terminator used was tfd from coli-phage fd (9).
Proceedings of the National Academy of Sciences, 1981
Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo.
Journal of Bacteriology, 1985
Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.
T7 RNA polymerase mutants with altered promoter specificities
Proceedings of the National Academy of Sciences, 1993
The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions-10 and-11 in the promoter. We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position. Surprisingly, most (13/19) sub