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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The N protein from bacteriophage lambda is a key regulator of transcription antitermination. It specifically recognizes a nascent mRNA stem loop termed boxB, enabling RNA polymerase to read through downstream terminators processively. The stacking interaction between Trp-18 of WT N protein and A7 of boxB RNA is crucial for efficient antitermination. Here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22). We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics. The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments. Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins.
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PMID:The RNA-protein complex: direct probing of the interfacial recognition dynamics and its correlation with biological functions. 1281 93

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.
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PMID:Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein. 1288 19

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2-aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi-step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half-life of around 1 s, which is followed by two slower steps occurring with a half-life in the range of minutes at 25 degrees C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi-step process. cAMP.CRP, which activates transcription at galP1, enhanced the rate-limiting step.
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PMID:Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. 1496 88

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.
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PMID:Evidence for the functional expression and pharmacological characterization of adenine receptors in native cells and tissues. 1560 13

N protein coded by phage lambda is a transcription factor that stimulates the antitermination activity of Escherichia coli RNA polymerase by binding specifically to the nascent RNA transcript at a stemloop structure called boxB. We use a new biophysical technique, involving the monitoring of the low energy circular dichroism spectra of 2-aminopurine residues site-specifically placed in the boxB RNA loop, to investigate this binding interaction. The low energy CD spectra of these 2-aminopurine probes reflect specific asymmetric interactions with adjacent nucleotide bases. Consequently, these CD spectra provide detailed and specific conformational information about the RNA chain at these chromophores that cannot be obtained from changes in the related fluorescence signals of these probes. CD changes were observed on binding the N peptide to boxB RNA that correspond to structural changes that had been previously seen by NMR, thus validating our experimental approach. The low energy CD method was then used to quantify the ordered and disordered states of the free hairpin loop and to show that a significant fraction of the boxB loop assumes a product-like structure in the absence of protein. A boxB derivative with an intact stem and a reduced concentration of ordered loop was identified and used to show that the extent of the reaction between protein and boxB depends on the concentration of structured loop in the RNA reactant population. This result has general implications for the conformational specificity of RNA-protein interactions.
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PMID:Low energy CD of RNA hairpin unveils a loop conformation required for lambdaN antitermination activity. 1603 60

To form a functional open complex, bacteriophage T7 RNA polymerase (RNAP) binds to its promoter DNA and induces DNA bending and opening. The objective of this study was to elucidate the temporal coupling in DNA binding, bending, and opening processes that occur during initiation. For this purpose, we conducted a combined measurement of stopped-flow fluorescence anisotropy, fluorescence resonance energy transfer (FRET), and 2-aminopurine fluorescence. Stopped-flow anisotropy measurements provided direct evidence of an intermediate resulting from rapid binding of the promoter to T7 RNA polymerase. Stopped-flow FRET measurements showed that promoter bending occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly bent. Similarly, stopped-flow 2-aminopurine fluorescence changes showed that promoter opening occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly melted. The indistinguishable observed rate constants of FRET and 2-aminopurine fluorescence changes indicate that DNA bending and opening processes are temporally coupled and these DNA conformational changes take place after the DNA binding step. The results in this paper are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which the promoter is both sharply bent and melted.
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PMID:Rapid binding of T7 RNA polymerase is followed by simultaneous bending and opening of the promoter DNA. 1660 62

The bacteriophage T7 elongation complex is an excellent model system in which to characterize the fundamental steps of transcription. We have formed functional elongation complexes, by mixing preassembled and RNA-primed DNA "bubble" constructs with T7 RNA polymerase and by initiating transcription at promoters, and have monitored the low-energy CD and fluorescence spectra of pairs of 2-aminopurine residues that have been inserted at defined sites within the DNA and RNA scaffold of the complex. In this way, we have been able to probe specific changes in the local conformations of the bases and base-pairs at these positions as the elongation complex goes through the various steps of the nucleotide addition cycle. The advantage of using pairs of 2-aminopurine residues, inserted at defined nucleic acid positions, as probes, is that the rest of the complex is spectrally "transparent" at wavelengths >300 nm. Thus, by combining CD and fluorescence measurements we obtain both structural and dynamic information that applies uniquely at each position within the functioning complex. In this way, we have mapped the details of steps central to transcription, including the formation and translocation of the transcription bubble, the formation and unwinding of the RNA-DNA hybrid, the passage of the nascent RNA through the exit channel of the polymerase, and the events of the template-controlled NTP selection process that controls transcriptional fidelity. This approach defines specific structural aspects of the elongation process under physiological conditions, and can be extended to examine other key aspects of transcriptional regulation, such as termination, editing, pausing, etc., that involve conformational rearrangements within the nucleic acid framework of the transcription complex.
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PMID:Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy. 1678 51

Fluorescent nucleobase analogues that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics, and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analogue. Responsive isosteric fluorescent pyrimidine analogues are, however, rare. Appending five-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogues with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial A-site, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.
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PMID:Fluorescent pyrimidine ribonucleotide: synthesis, enzymatic incorporation, and utilization. 1725 58

The distribution pattern of a 70 kDa cytokinin-binding protein (CBP70) was studied in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus). CBP70 was detected in crude protein extracts of all root zones and shoot parts by western blotting and by the sandwich ELISA (enzyme-linked immunosorbent assay) technique, using a pair of monoclonal anti-CBP70 antibodies cross-reacting with non-overlapping protein epitopes. The highest amount of CBP70 was found in the root meristem, which corresponds to the concentration in the meristem of zeatin, its riboside, nucleotide, and 9N-glucoside. CBP70 accumulation was also detected in other zones of cell division: in the root cap, shoot apex, and vascular tissues, suggesting involvement of the protein in the processes related to cell proliferation. This suggestion was also supported by CBP70 distribution in the root meristem: mitotically inactive cells of the quiescent centre did not contain a detectable amount of the protein. Stem cells adjoining the quiescent centre contained less CBP70 than their daughter cells. Using monoclonal antibodies against CBP70 for immunocytochemistry, the presence of the protein in the cytoplasm and its accumulation in nuclei and especially in nucleoli was demonstrated; such a pattern was observed in all cell types of seedlings. The subcellular distribution pattern of CBP70 was analysed by immunogold electron microscopy of the meristem and leaf cells; CBP70 was localized in the cytoplasm and nucleoplasm, and its highest concentration was detected in nucleoli. CBP70 was not detected in the vacuole and cell wall. In the RNA polymerase I model system, purified CBP70 mediated a trans-zeatin-dependent activation of transcription in vitro, and anti-CBP70 monoclonal antibodies blocked this activation. Other natural and synthetic physiologically active cytokinins also activated transcript elongation in the model system in the presence of CBP70. Adenine and inactive analogues of cytokinins had no such effects. These data suggest that CBP70 is a transcript elongation factor or a modulator of elongation factor activity specifically mediating a cytokinin-dependent regulation of transcription.
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PMID:Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings and its putative function. 1758 53

Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, whereas the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-Aminopurine probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency, and disrupt the upstream end of the hybrid as well as interfere with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently "lock" the polymerase to the nucleic acid scaffold and, thus, hold the RNA-DNA hybrid "in frame." At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, whereas hairpin-dependent pausing provides time for the process to go to completion.
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PMID:Direct spectroscopic study of reconstituted transcription complexes reveals that intrinsic termination is driven primarily by thermodynamic destabilization of the nucleic acid framework. 1807 Aug 78


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