Melting Of Promoter DNA Research Articles

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Regions 1.2 and 3.2 of the RNA Polymerase σ Subunit Promote DNA Melting and Attenuate Action of the Antibiotic Lipiarmycin

Initiation of RNA synthesis by bacterial RNA polymerase (RNAP) requires melting of promoter DNA, which is nucleated by the σ subunit during formation of the “open” promoter complex (RPo). The antibiotic lipiarmycin (Lpm) inhibits promoter melting by blocking access of the template DNA strand to the RNAP active-site cleft. Here we show that Escherichia coli RNAP holoenzymes containing either housekeeping σ70, with a deletion in the region 3.2, or the stationary phase σS subunits exhibited hypersensitivity to Lpm and increased cold sensitivity of RPo formation. Similar effects were produced by mutation located ~60Å away from the Lpm binding site within σ70 region 1.2, controlling −10 promoter element recognition. Our data suggested that template strand single-stranded DNA competes with Lpm for binding to RNAP and that σ70 regions 1.2 and 3.2 attenuate Lpm action by promoting DNA duplex opening.

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP–gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.

Transcription inactivation through local refolding of the RNA polymerase structure.

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

Threonine 429 of Escherichia coli σ 70 is a Key Participant in Promoter DNA Melting by RNA Polymerase

Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli σ 70 had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of σ 70: its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved − 11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the − 10 region. This model suggests an active role for RNA polymerase in disrupting the − 11 base pair, rather than just capturing the − 11A subsequent to spontaneous unpairing.

Strand Opening-deficient Escherichia coli RNA Polymerase Facilitates Investigation of Closed Complexes with Promoter DNA: EFFECTS OF DNA SEQUENCE AND TEMPERATURE

Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P(R) promoter of bacteriophage lambda to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the -35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37 degrees C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.

RETRACTED: Role of the Sigma Factor in Transcription Initiation in the Absence of Core RNA Polymerase

Sigma factors (sigmas) are bacterial transcription factors that bind core RNA polymerase (RNAP) and direct transcription initiation at cognate promoter sites. However, most of their functions have been investigated in the context of RNAP. This has made the exact function of sigma, and the importance of core RNAP in modulating sigma function, ambiguous. Here we identify a Bacillus subtilis mutant sigma(A) that is independently capable of specific binding and melting of the promoter DNA. Interestingly, specific and independent promoter binding of sigma is sufficient for the temperature- and Mg(2+)-independent melting of promoter DNA around the transcription start site, in contrast to the temperature- and Mg(2+)-dependent melting by RNAP around the promoter -10 element. Thus core RNAP is able to negatively modulate the sigma-initiated melting of the transcription start site and, by sensing the changes in temperature and Mg(2+) concentration, to regulate the efficiency of promoter -10 melting.

Mutational analysis of Escherichia coli heat shock transcription factor sigma 32 reveals similarities with sigma 70 in recognition of the -35 promoter element and differences in promoter DNA melting and -10 recognition.

Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor sigma32 rise greatly, resulting in the increased formation of the sigma32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on sigma32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving sigma32 expression from one plasmid and a beta-galactosidase reporter gene driven by the sigma32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on sigma32 activity. It was found that the recognition of the -35 region involves similar amino acid residues in regions 4.2 of E. coli sigma32 and sigma70. Three conserved amino acids in region 2.3 of sigma32 were found to be only marginally important in determining activity in vivo. Differences between sigma32 and sigma70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

Mechanistic Differences in Promoter DNA Melting by Thermus aquaticus and Escherichia coli RNA Polymerases

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations.

An Extended Winged Helix Domain in General Transcription Factor E/IIEα

Initiation of eukaryotic mRNA transcription requires melting of promoter DNA with the help of the general transcription factors TFIIE and TFIIH. Here we define a conserved and functionally essential N-terminal domain in TFE, the archaeal homolog of the large TFIIE subunit alpha. X-ray crystallography shows that this TFE domain adopts a winged helix-turn-helix (winged helix) fold, extended by specific alpha-helices at the N and C termini. Although the winged helix fold is often found in DNA-binding proteins, we show that TFE is not a typical DNA-binding winged helix protein, because its putative DNA-binding face shows a negatively charged groove and an unusually long wing, and because the domain lacks DNA-binding activity in vitro. The groove and a conserved hydrophobic surface patch on the additional N-terminal alpha-helix may, however, allow for interactions with other general transcription factors and RNA polymerase. Homology modeling shows that the TFE domain is conserved in TFIIE alpha, including the potential functional surfaces.

RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.

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, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.

β Subunit Residues 186–433 and 436–445 are Commonly Used by Eσ 54 and Eσ 70 RNA Polymerase for Open Promoter Complex Formation

During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript. Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level. The Escherichia coli RNAP holoenzyme is a six-subunit (α 2ββ′ωσ; Eσ) protein complex. The σ subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the β subunit have profound effects on promoter melting by Eσ 70. The σ 54 subunit is a representative of an unrelated class of the σ subunits. Here, we determined whether mutations in the β subunit that affect late stages of promoter complex formation by Eσ 70 also influence promoter complex formation by the enhancer-dependent Eσ 54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Eσ 54 suggest that during promoter complex formation by Eσ 54 and Eσ 70 a common set of β subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Eσ 70 and Eσ 54 thus proceed through a common pathway.

The intercalating β-hairpin of T7 RNA polymerase plays a role in promoter DNA melting and in stabilizing the melted DNA for efficient RNA synthesis

Phage T7 RNA polymerase contains within its single polypeptide all the elements for specific recognition and melting of its promoter DNA. Crystallographic studies indicate that a β-hairpin (230–245) with an intercalating valine residue plays a role in promoter opening. We mutated V237 to several amino acids, deleted five amino acid residues at the tip of the hairpin, and mutated E242 and D240 at the base of the hairpin to define the roles of the tip and base of the hairpin in DNA strand separation. The affinity of the hairpin mutants for the promoter DNA was not significantly affected. Stopped-flow kinetic studies showed that the bimolecular rate of DNA binding and the observed rate of pre-initiation open complex formation that corresponds to the sum of DNA opening and closing steps were within 20 to 40 % of the wild-type polymerase. Yet, most mutants showed a smaller amount of the pre-initiation open complex at equilibrium, indicating that the individual rates of promoter opening and closing steps were altered in the mutants. The base mutants, E242A and D240A, showed both a lower rate of promoter opening and a higher rate of promoter closing, suggesting their role in stabilization of the open complex. The V237D and the deletion mutant showed mainly a lower rate of promoter opening, suggesting that the tip of the hairpin may nucleate DNA opening. The defect in pre-initiation open complex formation affected downstream steps such as the rate of the first phosphodiester bond formation step, but did not affect significantly the apparent K d of initiating GTPs. We propose that D240 and E242 anchor the hairpin to the DNA and position the tip of the hairpin to allow V237 to intercalate and distort the DNA during open complex formation. The interactions of E242 and D240 with the upstream junction of the melted dsDNA promoter also align the template strand within the active site for efficient RNA synthesis.

Regulatory sequences in sigma 54 localise near the start of DNA melting

Transcription initiation by the enhancer-dependent σ 54 RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of σ 54 were used to map σ 54-DNA interaction sites. The two domains localise to form a centre over the −12 promoter region. The use of deletion mutants of σ 54 suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in σ 54-DNA contact and σ 54 conformation. The centre is close to the active site of the RNA polymerase and includes σ 54 regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

Sequence Determinants for the Recognition of the Fork Junction DNA Containing the −10 Region of Promoter DNA by E. coli RNA Polymerase

It has been recently suggested that E. coli RNA polymerase can specifically recognize a fork junction DNA structure, suggesting a possible role for such interaction in promoter DNA melting [Guo, Y., and Gralla, J. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11655-11660]. We have determined here quantitatively, using a site-specific binding assay, the effects of base substitutions within the conserved -10 hexamer in the context of a short fork junction DNA on binding to RNA polymerase. Adenine at position -11 and thymine at position -7 were found to be critical for sequence-specific recognition of the DNA. The identities of bases at positions -9 and -8 were found to be not important for the binding whereas replacement of bases at positions -12 and -10 had a mild negative effect on the binding affinity. It was found that for the binding of fork DNA to RNA polymerase, specific sequence recognition was more important than specific recognition of fork junction DNA structure. The pattern of relative importance of bases in the -10 region for binding RNA polymerase was generally consistent with the sequence conservation pattern observed in nature where positions -11 and -7 are the most conserved. Binding experiments with a series of adenine analogues at position -11 revealed that the N1 nitrogen of adenine was a critical determinant for the preference of the adenine at this position, suggesting a mechanism for the nucleation of promoter DNA melting initiation in which RNA polymerase destabilizes duplex DNA by directly competing with the thymine of the A-T base pair for hydrogen bonding to the N1 position of the -11 nontemplate strand adenine.

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled.

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

A Real-Time Fluorescence Method to Monitor the Melting of Duplex DNA during Transcription Initiation by RNA Polymerase

The melting of duplex DNA in the vicinity of the transcription start site is an essential step of transcription initiation. Here we describe a fluorescent promoter technique which allows the melting of promoter DNA to be observed in a real-time manner with high sensitivity. We have constructed a 114-bplacUV5 promoter fragment (−89 to +25) which contains a fluorescence probe in the region between the −10 consensus hexamer and the transcription start site. This region was chosen to incorporate a fluorescence probe as it undergoes strand separation subsequent to binding RNA polymerase (RNAP) (i.e., open complex formation). Upon mixing RNAP and fluorochrome-labeled promoter a time-dependent biphasic change in fluorescence was observed. The second slower component was shown to be due to the open complex by comparing the fluorescence data with the kinetics of open complex formation as measured by using alternative methods of open complex detection. The rate constants for open complex formation and dissociation were determined and found to be in excellent agreement with previously reported values. The techniques presented herein can generally be applied to other systems. Furthermore, this method will serve as an important research tool as well as it could be used in designing high-throughput assays involving transcription complexes.

The interaction between Bacillus subtilis sigma-A ( σA) factor and RNA polymerase with promoters

The P2 promoter from Bacillus subtilis sigma-A ( σ A) operon and the strong Ø29 phage G3b promoter were used to study their interactions with free σ A and with RNA polymerase holeenzymes (E σ A and E σ 70). No binding of free σ A to the tested promoters was observed, suggesting that the B subtilis free σ A does not bind promoter by itself for the initiation of RNA transcription. Different footprints of B subtilis RNA polymerase holoenzyme (E σ A) on the P2 and G3b promoters were detected. The footprint on the P2 promoter is mainly in the −10 downstream region of the bottom strand (noncoding strand) DNA and limited on the top strand (coding strand), whereas the footprints on both strands of the G3b promoter are very clear. These results suggest that the footprint regions of RNA polymerase on a promoter and the strength of its binding to the promoter depend on the properties of the specific promoter DNA sequence. It also suggests that the −10 and its downstream regions are more important than the −35 region for the formation of the E σ A-P2 promoter open complex. Footprints of B subtilis E σ A and E coli Eσ 70 on the same G3b promoter are very similar on the top strand but different on the bottom strand, with the footprint being about 17 bases wider (−4 to +13) in the case of E coli E σ 70. Since this region contains most of the basesinvolved in promoter DNA melting, we suggest that E coli and B subtilis RNA polymerases have different efficiency in forming the open complex with heterologous promoter DNA during initiation of transcription.

Activator-dependent preinduction binding of .sigma.-70 RNA polymerase at the metal-regulated mer promoter

Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion [Hg(II)] and activates it in the presence of Hg(II). In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD). Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR. Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry. Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex.

KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo

The reagent potassium permanganate is used to probe lac transcription complexes by primer extension-probing analysis. A series of strongly hyperreactive bands, corresponding to the known melted region, is observed when open complexes are formed in vitro. A nearly identical pattern occurs in vivo, the signal intensity of which increases when open complexes are trapped with rifampicin. Quantitative comparison of the signal intensity obtained under steady-state conditions with that obtained in the presence of rifampicin indicates that transcription from each of three lac promoter variants is limited in vivo principally by the slow rate of open complex formation. The slow-start lac L8:UV5 promoter is also limited somewhat by slow RNA chain initiation. Slightly different patterns of KMnO4 reactivity at each promoter variant in the absence of RNA polymerase in vitro suggest that DNA sequence dictates the ultimate pattern of melting, with the polymerase acting principally to stabilize the melted state specified by the DNA sequence.