Quantitative Determination of Oct4-Sox2 Heterodimer Formation with Nanog Promoter Element (original) (raw)
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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.
Promoter Recognition As Measured by Binding of Polymerase to Nontemplate Strand Oligonucleotide
Science, 1997
In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase. As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding. Specific interaction between RNA polymerase and the promoter in Escherichia coli was detected and quantified as the binding affinity of nontemplate oligonucleotide sequences. The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.
The mechanism of DNA replication primer synthesis by RNA polymerase
Nature, 2006
RNA primers for DNA replication are usually synthesized by specialized enzymes, the primases 1 . However, some replication systems have evolved to use cellular DNA-dependent RNA polymerase for primer synthesis 1,2 . The main requirement for the replication primer, an exposed RNA 3 0 end annealed to the DNA template, is not compatible with known conformations of the transcription elongation complex 3 , raising a question of how the priming is achieved. Here we show that a previously unrecognized kind of transcription complex is formed during RNA polymerasecatalysed synthesis of the M13 bacteriophage replication primer. The complex contains an overextended RNA-DNA hybrid bound in the RNA-polymerase trough that is normally occupied by downstream double-stranded DNA, thus leaving the 3 0 end of the RNA available for interaction with DNA polymerase. Transcription complexes with similar topology may prime the replication of other bacterial mobile elements and may regulate transcription elongation under conditions that favour the formation of an extended RNA-DNA hybrid.
Identification of a minimal binding element within the T7 RNA polymerase promoter
Journal of Molecular Biology, 1997
The T7 RNA polymerase promoter has been proposed to contain two domains: the binding region upstream of position À5 is recognized through apparently traditional duplex contacts, while the catalytic domain downstream of position À5 is bound in a melted con®guration. This model is tested by following polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence. The increase in the¯uorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations, and allows the determination of both increases and decreases in the dissociation constant. The best ®t value of K d 4.0 nM for the native promoter is in good agreement with previous¯uorescence and steady state measurements. Deletion of the downstream DNA up to position À1 or to position À5 leads to a ®vefold increase in binding, while further sequential single-base deletions upstream result in 20 and 500-fold decreases in binding. These results indicate that the (duplex) region of the promoter upstream of and including position À5 is both necessary and suf®cient for tight binding, and represents the core binding element of the promoter. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. We also show that the presence of magnesium is necessary for optimal binding, but not for speci®c enzyme-promoter complex formation, and we propose that magnesium is not required for melting of the promoter.
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.
Journal of molecular biology, 2006
DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide-(NTP) and deoxyribonucleotidetriphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2′-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2′-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano-and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.