E. coli RNA polymerase interacts homologously with two different promoters (original) (raw)
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Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter
Proceedings of the National Academy of Sciences, 1979
I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine. In the lac promoter most of these contacts lie in three regions. Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase. Which features of a promoter direct RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) to initiate RNA synthesis? DNA sequence determination of promoters and promoter mutants provides a partial answer to this question. When the sequences of promoters are compared, two regions of prominent homology have been observed: the "Pribnow box" and the "-35 region" located about 10 and 35 base pairs, respectively, upstream from the start site of . Furthermore, almost all promoter mutations are located within these regions (5-12).
RNA polymerase-promoter interactions: the comings and goings of RNA polymerase
Journal of bacteriology, 1998
1. Amouyal, M., and H. Buc. 1987. Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. J. Mol. Biol. 195:795-808. 2. Ayers, D. G., D. T. Auble, and P. L. deHaseth. 1989. Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation. J. Mol. Biol. 207:749-756. 3. Barkley, M. D. 1981. Salt dependence of the kinetics of the lac repressoroperator interaction: role of nonoperator deoxyribonucleic acid in the association reaction. Biochemistry 20:3833-3842. 4. Barne, K. A., J. A. Bown, S. J. W. Busby, and S. D. Minchin. 1997. Region 2.5 of the Escherichia coli RNA polymerase 70 subunit is responsible for the recognition of the "extended Ϫ10" motif at promoters. EMBO J. 16:4034-4040. 5. Blatter, E. E., W. Ross, H. Tang, R. L. Gourse, and R. H. Ebright. 1994. Domain organization of RNA polymerase ␣ subunit: C-terminal 85 amino acids constitute an independently folded domain capable of dimerization and DNA binding. Cell 78:889-896. 6. Brunner, M., and H. Bujard. 1987. Promoter recognition and promoter strength in the Escherichia coli system. EMBO J. 6:3139-3144. 7. Buckle, M., and H. Buc. 1989. Fine mapping of DNA single stranded regions using base-specific chemical probes: study of an open complex formed between RNA polymerase and the lac UV5 promoter. Biochemistry 28:4388-4396. 8. Burgess, R., A. Travers, J. J. Dunn, and E. K. F. Bautz. 1969. Factor stimulating transcription by RNA polymerase. Nature 221:43-46. 9. Carpousis, A. J., and J. D. Gralla. 1980. Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry 19:3245-3253. 10. Chen, Y.-F., and J. D. Helmann. 1997. DNA melting at the Bacillus subtilis flagellin promoter nucleates near Ϫ10 and expands unidirectionally. J. Mol. Biol. 267:47-59. 11. Craig, M. L., W.-C. Suh, and M. T. Record, Jr. 1995. HO⅐ and DNase I probing of E 70 RNA polymerase-PR promoter open complexes: Mg 2ϩ binding and its structural consequences at the transcription start site. Biochemistry 34:15624-15632. 11a.Craig, M. L., •. •. Tsodikov, •. •. Saecker, and M. T. Record, Jr. Unpublished data. 12. Darst, S. A., E. W. Kubalek, and R. D. Kornberg. 1989. Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography. Nature 340:730-732. 12a.deHaseth, P. L., et al. Unpublished data. 13. deHaseth, P. L., and J. D. Helmann. 1995. Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced
Journal of Biological Chemistry
Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed. Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition. Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role. Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.
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.
Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase
The EMBO Journal, 1991
Communicated by W.Zillig Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.
Nucleic Acids Research, 1995
Footprinting data for 33 open promoter complexes with Escherichia coli RNA polymerase, as well as 17 ternary complexes with different regulators, have been compiled using a computer program FUTPR. The typical and individual properties of their structural organization are analyzed. Promoters are subgrouped according to the extent of the polymerase contact area. A set of alternative sequence elements that could be responsible for RNA polymerase attachment in different promoter groups is suggested on the basis of their sequence homology near the hyperreactive sites. The model of alternative pathways used for promoter activation Is discussed.
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.
Altered promoter recognition by mutant forms of the ?70 subunit of Escherichia coli RNA polymerase*1
J Mol Biol, 1989
We have systematically assayed the in viva promoter recognition properties of 13 mutations in rpoD, the gene that encodes the 0" subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of c" and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of cr", which is similar to the helix-turnhelix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the-35 region, and that a sequence internal to a", in a region which is highly conserved among Q factors, recognizes the-10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the-35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effects on promoter recognition.
RNA-polymerase binding at the promoters of the rRNA genes of Escherichia coli
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1980
The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis of polymerase binding, transcription initiation and nitrocellulose filtration of RNA-polymerase-DNA complexes, using restriction endonuclease generated fragments of recombinant plasmids and a transducing phage. The following observations have been made: 1. Two transcription initiation sites have been located approximately 200 and 300 base pairs upstream from the beginning of the sequence coding for mature 16 S rRNA. 2. Polymerase binding at these sites can be observed electronmicroscopically and a 360 base-pair fragment containing these sites binds to nitrocellulose in the presence of RNA-polymerase. This complex dissociates even at moderately high (0.1-0.2 M) salt concentrations. Although transcription initiation is reported to be more frequent at the first of these sites, the binding is much stronger at the second site. 3. In the case of the rrnD gene, BamHI cleaves a few base pairs upstream from the first transcription start site. This cleavage destroys polymerase binding at this site but does not influence binding at the second site. 4. At higher polymerase/DNA ratio four weak but distinct and regularly spaced binding sites can be observed preceding the two initiation sites at approximately 1000, 820, 640 and 440 base pairs before the mature 16 S rRNA sequence. 5. An extremely strong binding site is located about 1300 base pairs upstream from the beginning of the 16 S rRNA sequence. Very little (if any) initiation occurs at this site. The possibility is discussed that the noninitiating binding sites preceding the two transcription start points might functionally belong to the promoter region.