Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5 (original) (raw)
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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).
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.
Proceedings of the National Academy of Sciences of the United States of America, 1979
A transducing phage has been isolated which codes for the a subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage X vector that is capable of forming Iyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the a gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and o (rpoD) whereas those containing the Sac I fragment complement only a. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/ translation system suggest that a Sac I site separates the promoter for a from the a structural gene. The direction of transcription of a was determined to be clockwise with respect to the E. coli genetic map. Escherichia coli RNA polymerase is a multisubunit enzyme composed of a, 13, f3', and oa subunits. The enzyme is found in two forms: as holoenzyme (a2/3f'), capable of selective DNA
Continuous in vitro Evolution of Bacteriophage RNA Polymerase Promoters
Rapid in vitro evolution of bacteriophage T7, T3, and SP6 R N A polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent R N A polymerase. Replication involves the synthesis of both R N A and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of R N A transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce R N A transcripts. Furthermore, each R N A transcript encodes the R N A polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7,
Proceedings of The National Academy of Sciences, 1985
The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.
Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters
Journal of Biological Chemistry, 1991
The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and-12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerasepromoter complexes.
Microbiology (Reading, England), 2000
The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P...
Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA
Journal of Molecular Biology, 1998
The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter speci®city subunit, s 70 , with high af®nity and inhibits its transcription activity. AsiA binds to s 70 primarily through an interaction with s 70 conserved region 4.2, which has also been implicated in sequence-speci®c recognition of the À35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and s 70 and thus does not inhibit s 70 activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two À10/À35 type promoters and two``extended À10'' promoters. Our results indicate that the binding of AsiA to s 70 and the interaction of s 70 region 4.2 with the À35 consensus promoter element of À10/À35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended À10 promoters, which lack a À35 consensus element and do not require s 70 conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP s 70 holoenzyme transcription at À10/ À35 promoters by interfering with the required interaction between s 70 conserved region 4.2 and the À35 consensus promoter element.
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.