Open Promoter Complex Research Articles

Evidence of DNA bending in transcription complexes imaged by scanning force microscopy.

Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope. The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template. The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript. This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.

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.

Central domain of the positive control protein NifA and its role in transcriptional activation

The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing σ 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E σ 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.

Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli

The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the −35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated. We can show, using the abortive initiation assay, that the prodominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant K B) while the isomerisation rate ( E f) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterization of sequence determinants important for stringent regulation and growth rate control. Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.

Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function

A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.

Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation.

Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.

In vivo studies on the interaction of RNA polymerase-σ 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters : The role of NifA in the formation of an open promoter complex

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor σ 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase- σ 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed σ 54-dependent methylation protection of guanine residues at −14, −25 and −26, the most conserved nucleotides characteristic of σ 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from − 15 to −17 showed σ 54-dependent methylation protection of guanines −13, −24 and −25. The enhanced interaction of the RNA polymerase- σ 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO 4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the σ 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to −10 reactive towards KMnO 4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of σ 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase- σ 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.

RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.

Repression of the λ pcin promoter by integrative host factor

The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda. The pcin promoter transcribes the cI repressor gene constitutively. λ cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF). λ cI − cin-1 grows normally in IHF − mutants, indicating that repressor overproduction from pcin blocks lytic growth. The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription. We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells. In vitro studies show that IHF directly inhibits transcription initiation at pcin. Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter. RNA polymerase bound in an open promoter complex is resistant to IHF. We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure.

Rotational Orientation of Upstream Curved DNA Affects Promoter Function in Bacillus subtilis

The Alu156 promoter isolated from the Bacillus subtilis bacteriophage SP82 is dependent on curved DNA upstream of the -35 region for efficient function. Short DNA insertions of 6-29 base pairs were used to simultaneously change the linear placement and rotational orientation of this curved DNA relative to the -35 region. When these mutant promoters were analyzed in vivo using transcriptional fusions with a chloramphenicol acetyltransferase gene, changes in the rotational orientation of the curved DNA correlated with changes in promoter function. The most efficient mutant promoters contained insertions of 11 and 21 base pairs, and insertions of 15 and 25 base pairs resulted in the least efficient mutant promoters. The importance of the proper rotational alignment of the curved DNA to promoter activity was also observed in vitro at the level of transcription of RNA polymerase binding. Based on the electrophoretic mobilities of DNA fragments containing the various insertion mutant promoters, there was a second region of curved DNA downstream of the insertion point. The findings are consistent with the idea that the curved DNA deflects the helix back toward the promoter-bound RNA polymerase molecule to allow the enzyme to interact directly with upstream DNA. These interactions are proposed to structure the DNA for the formation of the open promoter complex.

Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex

Characterization of ternary complexes containing an Escherichia coli lac promoter DNA fragment, CAP protein and RNA polymerase, separated on non-denaturing polyacrylamide gels and footprinted in the gel slice, reveals a striking stabilization of CAP against dissociation in the open complex, compared to the CAP-DNA complex lacking RNA polymerase. The stabilization is lost when half a helical turn of DNA is inserted between CAP and polymerase sites, but is partially restored with an 11 base-pair insert; stimulation of transcription parallels the stabilization effect. This behavior suggests a direct protein-protein interaction. Comparison of initiation kinetics for wild-type and a mutant in which the P2 promoter has been inactivated shows that CAP both strengthens binding in the closed complex and accelerates isomerization to the open complex; the latter effect accounts for the bulk of the observed transcriptional activation.

Micrococcal nuclease as a probe for bound and distorted DNA in lac transcription and repression complexes.

Micrococcal nuclease (MNase) is used to probe the structure of transcription and repression complexes at the lac regulatory region in vitro. Both the lac operator, 01, and the pseudo-operator, 03, are found to be protected from MNase digestion by the lac repressor on supercoiled DNA, and hypersensitive sites appear on both strands around nucleotide (nt) -26 between 01 and 03. This hyperreactive site is coincident with the site of the DNA kink shown previously to form within a loop caused by simultaneous repressor binding to 01 and 03. MNase hypersites are also observed both upstream from cAMP receptor protein (CRP) and downstream from bound RNA polymerase in open promoter complexes. In both open and closed complexes the binding of polymerase partially protects the backbone from MNase attack. Catabolite activator protein is shown to be required for both closed and open complex formation. Taken together with previous footprinting data, the results suggest that lac transcription complexes involve DNA bent towards a protein core consisting of RNA polymerase and catabolite activator protein.

Uranyl mediated photofootprinting reveals strongE.coliRNA polymerase-DNA backbone contacts in the +10 region of theDeoP1 promoter open complex

Employing a newly developed uranyl photofootprinting technique (Nielsen et al. (1988) FEBS Lett. 235, 122), we have analyzed the structure of the E. coli RNA polymerase deoP1 promoter open complex. The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region. These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region. The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded. The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region.

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters.

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein.

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.

Interactions of the bacteriophage T4 gene 55 product with Escherichia coli RNA polymerase. Competition with Escherichia coli sigma 70 and release from late T4 transcription complexes following initiation.

The bacteriophage T4 gene 55 protein endows Escherichia coli RNA polymerase core with the ability to recognize T4 late promoters. In these experiments, we show that T4 gene product (gp) 55, like the major E. coli RNA polymerase initiation subunit sigma 70, is released from elongating transcription complexes and is thus capable of cycling among molecules of RNA polymerase core. We also show that, paradoxically, sigma 70 is able to displace T4 gp55 from its site of binding to E. coli RNA polymerase core. After T4 infection, the core enzyme is ADP-ribosylated in each alpha subunit, and a T4-coded 11.4-kDa protein (rpbA) binds tightly to it; these modifications do not save gp55 from displacement by sigma 70. However, open T4 late promoter complexes, containing gp55, are resistant to attack by sigma 70.

A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein.

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.

Supercoiling response of the lac p s promoter in vitro

The rate of open promoter complex formation was measured on lac p s promoter DNA templates differing in negative superhelicity. The templates ranged from fully relaxed to those with numbers of superhelical turns exceeding that of form I plasmid DNA. The observed transcription response had two clearly distinguished phases: an initial rapid rise in rate followed eventually by a precipitous inhibition. The stimulation phase involved a nearly 40-fold increase in rate, which peaks at superhelical densities near that of isolated form I plasmid DNA. The introduction of more negative superhelical turns leads to inhibition. The magnitude of the response and the observation of both increases and decreases suggest that minor differences in superhelicity in vivo could lead to significant increases or decreases in transcription rate. The increase in rate was found to be directly proportional to the free energy of supercoiling; that is, to the square of the superhelical density. We suggest that the energy may be used both for enhanced DNA melting and for changes in DNA structure that alter the helical “face” with which RNA polymerase must interact. A quantitative method is presented that allows simple estimation of differences in the supercoiling response among promoters, both in the presence and in the absence of added factors.