DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study (original) (raw)
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Identification and analysis of 'extended -10' promoters in Escherichia coli
Nucleic Acids Research, 2003
We have compiled and aligned the DNA sequences of 554 promoter regions from Escherichia coli and analysed the alignment for sequence similarities. We have focused on the similarities and differences between promoters that either do or do not contain an extended ±10 element. The distribution of ±10 and ±35 hexamer element sequences, the range of spacer lengths between these elements and the frequencies of occurrence of different nucleotides, dinucleotides and trinucleotides were investigated. Extended ±10 promoters, which contain a 5¢-TG-3¢ element, tend to have longer spacer lengths than promoters that do not. They also tend to show fewer matches to the consensus ±35 hexamer element and contain short runs of T residues in the spacer region. We have shown experimentally that the extended ±10 5¢-TG-3¢ motif contributes to promoter activity at seven different promoters. The importance of the motif at different promoters is dependent on the sequence of other promoter elements.
Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures
The EMBO Journal vol.5 no. 11 pp.2987-2994, 1996
The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy. Up to 75-fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5. Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence. By contrast, a synthetic 'consensus promoter' belongs to the less efficient signals. Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that infonrnation outside of the 'classical' promoter region contributes to promoter activity. Key words: E. coli promoters/in vivo strength/alternate structures Introduction Despite a wealth of information on the structure of Escherichia coli promoters, the topography of RNA polymerase/promoter complexes and on the processes governing the onset of specific transcription (for review, see Rosenberg and Court, 1979), our understanding of structure/function relationships of E. coli promoters still remains unsatisfactory. The steadily increasing number of elucidated promoter sequences has allowed the extensive study of structural homologies and refined 'consensus se-quences' were proposed (for review, see Hawley and McClure, 1983). So far, however, such model sequences were of little predictive value, whenever functional parameters of an individual promoter were to be derived from structural information alone. Quantitative information on the function of defined promoter sequences is obviously required for a better understanding of how the complex functional programme of a promoter is stored in a DNA sequence. Here we describe an experimental system for the accurate determination of promoter strength in vivo and in vitro. Promoter activities are measured by monitoring RNA synthesis in relation to an internal standard. Thus, the results are independent of trans-lational effects and of gene dosage. Differences in mRNA half-life can be taken into account. Fourteen promoters were char
SN Applied Sciences, 2021
The gene transcription of bacteria starts with a promoter sequence being recognized by a transcription factor found in the RNAP enzyme, this process is assisted through the conservation of nucleotides as well as other factors governing these intergenic regions. Faced with this, the coding of genetic information into physical aspects of the DNA such as enthalpy, stability, and base-pair stacking could suggest promoter activity as well as protrude differentiation of promoter and non-promoter data. In this work, a total of 3131 promoter sequences associated to six different sigma factors in the bacterium E. coli were converted into numeric attributes, a strong set of control sequences referring to a shuffled version of the original sequences as well as coding regions is provided. Then, the parameterized genetic information was normalized, exhaustively analyzed through statistical tests. The results suggest that strong signals in the promoter sequences match the binding site of transcri...
Nucleic Acids Research, 1991
A series of E. coli promoters made of the consensus -35 and -10 hexamers separated by 17 bp spacer with variously located bending dTn dAn, n = 5 or 6, sequences was constructed and cloned into the plasmid pDS3. Electrophoretic gel mobilities of restriction fragments containing these promoters correlated with the number of the T tracts encoded in the promoter sequences. The open complexes formed by E. coli RNA polymerase on promoters containing the T-(-34... -38) tract exhibited gel retardation indicative of their different gross geometry. The strength of these promoters measured in vivo in relation to an internal transcriptional standard was shown to be significantly lower than that of the group without the T5(-34...-38) tract. Within both these groups the promoters with two T6 tracts in the spacer, aligned in phase with the B-DNA helix repeat, had lower transcriptional activity, while the T6 tract encoded in the -7... -2 promoter region apparently had no influence on the strength of the respective promoters.
Biochemistry, 1993
Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region. These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence. Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP elementcontaining promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase. We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperaturesensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes. Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction.
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.
[Distribution and functional significance of A/T tracts in promotor sequences of Escherichia coli]
Molekuliarnaia biologiia
Distribution of the A/T tracts described in earlier publications in the region extending from nucleotide -250 to +150 relative to the transcription initiation site of gene transcribed regions adjacent to promoter was studied. Upstream of the -35 region a succession of A/T tracts was discovered distributed at a shorter distance one from another than in analogous elements of the transcribed region (1 and 1.5 helix turns, respectively). Such a positional dependence suggests different functional manifestation of A/T tracts at different transcription steps. Single initiation using the T7D promoter mutant derivatives devoid of A/T tracts in two critical positions, +41 and , yielded shortened products of the corresponding length. One might speculate that such elements adjacent to promoter region play a significant role in transcription complex functioning.
Anatomy of Escherichia coli 70 promoters
Nucleic Acids Research, 2007
Information theory was used to build a promoter model that accounts for the À10, the À35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base À7, rather than À11, of the À10 may be flipping to initiate transcription. We found that the sequence conservation of s 70 binding sites is 6.5 ± 0.1 bits. Some promoters lack a À35 region, but have a 6.7 ± 0.2 bit extended À10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended À10 binding and the À35 suggest that the flexible bipartite s factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5 0 mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNAbending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the À35, so bending allows distal activators to reach the polymerase.
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).
Analysis of Oligonucleotide Composition in Dna of E. Coli Genome and Promoter Sites
Summary Motivation: Recent identification of some noncanonical determinants in E. coli promoters has opened a field for a search of new candidates for this role. As suggested and shown here, a large- scale comparative analysis of oligonucleotide composition of the complete sequence of E. coli genome and its promoter sites has much potential for yielding information about all kinds of nucleotide blocks preferred by promoters that might contribute to promoter recognition. Results: The content of all kinds of hexa-, hepta- and octonucleotides in the complete E. coli genome and in 359 promoters was determined. A nonrandom distribution of oligonucleotides was found both in the complete genome and in the promoters. The complete genome and the promoters were shown to differ essentially in their oligonucleotide sets. 542 different hexanucleotides are twofold more frequent in the promoters than in the chromosome. Hexanucleotides most preferred by promoters are discussed as possible candidate...