Quantitative Determination of Oct4-Sox2 Heterodimer Formation with Nanog Promoter Element (original) (raw)
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 Unpublished data. 12.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.
Indirect read-out of the promoter DNA by RNA polymerase in the closed complex
Nucleic Acids Research, 2013
Transcription is initiated when RNA polymerase recognizes the duplex promoter DNA in the closed complex. Due to its transient nature, the closed complex has not been well characterized. How the initial promoter recognition occurs may offer important clues to regulation of transcription initiation. In this article, we have carried out single-base pair substitution experiments on two Escherichia coli promoters belonging to two different classes, the À35 and the extended À10, under conditions which stabilize the closed complex. Single-base pair substitution experiments indicate modest base-specific effects on the stability of the closed complex of both promoters. Mutations of base pairs in the À10 region affect the closed complexes of two promoters differently, suggesting different modes of interaction of the RNA polymerase and the promoter in the two closed complexes. Two residues on p 70 which have been suggested to play important role in promoter recognition, Q437 and R436, were mutated and found to have different effects on the closedcomplex stability. DNA circular dichroism (CD) and FRET suggest that the promoter DNA in the closed complex is distorted. Modeling suggests two different orientations of the recognition helix of the RNA polymerase in the closed complex. We propose that the RNA polymerase recognizes the sequence dependent conformation of the promoter DNA in the closed complex.
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.
Biochemistry, 2002
Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position -11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the -11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the -11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position -11 has no major effect on the accessibility of DNA to DNase I or KMnO 4 in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at -11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparinresistant complex with RNA polymerase. These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.
Journal of Molecular Biology, 2009
Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P R and P RM promoters of bacteriophage λ have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P R [Rivetti, C., . Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464-4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from − 36 to − 59 and from − 80 to − 100. Likewise, RPos formed at P RM showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of αsubunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P R promoter. We find, however, that the frequency of DNA templates with both P R and P RM occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that α carboxyterminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P R and P RM is proposed.
E. coli RNA polymerase interacts homologously with two different promoters
Cell, 1980
We present and review experiments that identify points of close approach of the RNA polymerase to two promoters, lac UV5 and T7 A3. We identify the contacts to the phosphates along the DNA backbone, to the N7s of guanines in the major groove and the N3s of adenines in the minor groove, and to the methyl groups of thymines. These contacts to the two promoters are strikingly homologous in space, as shown on three-dimensional models, and identify major regions of interactions lying on one side of the DNA molecule (at-35 and-16), as well as further areas extending through the Pribnow box. Both promoters are unwound similarly by the polymerase, across a region of about twelve bases extending from the middle of the Pribnow box to just beyond the RNA start site. We discuss the areas of interaction in the context of promoter homologies and promoter mutations. The disposition of the contacts in space suggests a model for the pathway along which the RNA polymerase binds to promoters.