Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures (original) (raw)

Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal (promoter efficiency/RNA synthesis/RNA polymerase/gene expression

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. §1734 solely to indicate this fact.

Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process.

The EMBO Journal, 1986

After binding to a promoter Eschewchia coli RNA polymerase is in contact with a region of about 70 bp. Around 20 bp of this sequence are transcribed. Information encoded within this transcribed region is involved in late steps of the functional program of a promoter. By changing such 'downstream' sequences promoter strength in vivo can be varied more than 10-fold. By contrast, information for early steps of the promoter program such as recognition by the enzyme and formation of a stable complex resides in a central core region of about 35 bp. Our data show that the strength of a promoter can be limited at different levels of the overall process. Consequently promoters of identical strength can exhibit different structures due to an alternate optimization of their program.

Promoters Recognized by Escherichia coli RNA Polymerase Selected by Function: Highly Efficient Promoters from Bacteriophage T5 Downloaded from

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).

Promoter search and strength of a promoter: two important means for regulation of gene expression inEscherichia coli

Journal of Biosciences, 1993

Search for a promoter element by RNA polymerase from the extremely large DNA base sequence is thought to be the slowest and rate-determining for the regulation of transcription process. Few direct experiments we described here which have tried to follow the mechanistic implications of this promoter search. However, once the promoter is located, transcription complex, constituting mainly the RNA polymerase molecule and few transcription factors has to unidirectionally clear the promoter and elongate the RNA chain through a series of steps which altogether define the initiation of transcription process. Thus, it appears that the promoter sequence acts as a trap for RNA polymerase associated with a large binding constant, although to clear the promoter and to elongate the transcript such energy barrier has to be overcome. Topological state of the DNA, particularly in the neighbourhood of the promoter plays an important role in the energetics of the whole process.

Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes

Nucleic Acids Research, 2005

During the process of transcription, RNA polymerase can exactly locate a promoter sequence in the complex maze of a genome. Several experimental studies and computational analyses have shown that the promoter sequences apparently possess some special properties, such as unusual DNA structures and low stability, which make them distinct from the rest of the genome. But most of these studies have been carried out on a particular set of promoter sequences or on promoter sequences from similar organisms. To examine whether the promoters from a wide variety of organisms share these special properties, we have carried out an analysis of sets of promoters from bacteria, vertebrates and plants. These promoters were analyzed with respect to the prediction of three different properties, such as DNA curvature, bendability and stability, which are relevant to transcription. All the promoter sequences are predicted to share certain features, such as stability and bendability profiles, but there are significant differences in DNA curvature profiles and nucleotide composition between the different organisms. These similarities and differences are correlated with some of the known facts about transcription process in the promoters from the three groups of organisms.

Promoter recognition by Escherichia coli RNA polymerase

Journal of Molecular Biology, 1989

The available evidence suggests that during the process of formation of a functional or "open" complex at a promoter, Escherichia coli RNA polymerase transiently realigns the two contacted regions of the promoter, thus stressing the intervening spacer DNA. We tested the possibility that this process plays an active role in the formation of an open complex. Two series of promoters were examined: one with spacer DNAs of 15 to 19 basepairs and a derivative for which the promoters additionally contained a one-base gap in the spacer, so as to relieve any stress imposed on the DNA. Consistent with an active role for the stressed DNA in driving open complex formation, we have found that for promoters with a 17-base-pair spacer, the presence of a gap leads to a delay in the formation of an open complex, at a step subsequent to the initial binding of RNA polymerase to the promoter. The results with the other gapped promoters rule out direct binding of RNA polymerase to the region of the gap and indicate an increased flexibility in the gapped DNA. As not all observations with the spacer length series of gapped and ungapped promoters can be interpreted in terms of an active role of the spacer DNA without additional assumptions, such a role must still be considered tentative.

Proximal transcribed regions of bacterial promoters have a non-random distribution of A/T tracts

Nucleic Acids Research, 1999

Promoter sequences of Escherichia coli were compiled and their transcribed regions characterized by site-specific cluster analysis. Here we report that transcribed regions contain a non-random distribution of A/T tracts with strongly preferred positions at 6 ± ± ± ± 3, 23 ± ± ± ± 3, 40 ± ± ± ± 2 and 56 ± ± ± ± 2. The maxima of this distribution follow an unusual periodicity (~17 bp) and are in phase with important promoter elements involved in interaction with RNA polymerase, while the value of periodicity numerically fits the spacer length between the canonical -35 and -10 elements. The possible functional significance of this newly described feature is discussed in the context of promoter clearance and transcription pausing.

The variation of promoter strength in different gene contexts

2020

BackgroundPromoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. There have been many effortless studies searching the best promoter for biological application. However, whether promoter strengths stay unchanged in different gene contexts remains unknown.ResultsSix consecutive promoters at different strength levels were used to construct six different versions of plasmid backbone pTH1227, followed by inserted genes encoding two polymer-producing enzymes. Some of promoter strengths in the presence of inserted sequences did not correspond to the reported strengths in a previous study. When removing the inserted sequences, the strengths of these promoters returned to their reported strengths. These changes were further confirmed to occur at transcriptional levels. Polymer production using our newly constructed plasmids showed polymer accumulation levels relatively corresponding to the promoter strengths reported in our study.Conclus...