Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination.
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Passage of E. coli RNA polymerase through an intrinsic transcription terminator, which encodes an RNA hairpin followed by a stretch of uridine residues, results in quick dissociation of the elongation complex. We show that folding of the hairpin disrupts the three upstream base pairs of the 8 bp RNA:DNA hybrid, a major stability determinant in the complex. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. During termination, the hairpin does not directly compete for base pairing with the 8 bp hybrid. Thus, melting of the hybrid seems to result from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Our results suggest that a similar mechanism disrupts elongation complexes of yeast RNA polymerase II in vitro. (+info)
Mutational and functional analysis of a segment of the sigma family bacteriophage T4 late promoter recognition protein gp55.
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Bacteriophage T4 late promoters, which consist of a simple 8-base pair TATA box, are recognized by the gene 55 protein (gp55), a small, highly diverged member of the sigma family proteins that replaces sigma(70) during the final phase of the T4 multiplication cycle. A 16-amino acid segment of gp55 that is proposed to be homologous to the sigma(70) region 2.2 has been subjected to alanine scanning and other mutagenesis. The corresponding proteins have been examined in vitro for binding to Escherichia coli RNA polymerase core enzyme and for the ability to generate accurately initiating basal as well as sliding clamp-activated T4 late transcription. Mutations in the amino acid 68-83 segment of gp55 generate a wide range of effects on these functions. The changes are interpreted in terms of the multiple steps of involvement of gp55, like other sigma proteins, in transcription. Effects of mutations on RNA polymerase core binding are consistent with the previously proposed homology of amino acids 68-82 of gp55 with sigma(70) region 2.2 and the recently determined structures of the Thermus thermophilus and Thermus aquaticus sigma(70)-RNA polymerase holoenzymes. (+info)
Thermoirreversible and thermoreversible promoter opening by two Escherichia coli RNA polymerase holoenzymes.
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Promoter opening, in which the complementary DNA strands separate around the transcriptional start site, is generally thermoreversible. An exceptional case of thermoirreversible opening of the T4 late promoter has been analyzed by KMnO4 footprinting and transcription. T4 late promoters, which consist of an 8-base pair (bp) TATA box "-10" element, are recognized by the small, phage-encoded, highly diverged sigma-family initiation subunit gp55. The T4 late promoter only opens above 15-20 degrees C, but once it has been formed remains open and transcriptionally active for days at -0.5 degrees C. The low temperature-trapped open complex and its isothermally formed state are shown to be structurally distinctive. Two "extended -10" sigma 70 promoters, which, like the T4 late promoter, lack "-35" sites, have been subjected to a comparative analysis: the T4 middle promoter PrIIB2 opens and closes thermoreversibly under conditions of basal and MotA- and AsiA-activated transcription. The open galP1 promoter complex, whose transcription bubble is very AT-rich, also closes reversibly upon shift to -0.5 degrees C, but more slowly than does the rIIB2 promoter. Formation of a trapped-open low temperature state of the promoter complex appears to be a singular property of gp55-RNA polymerase holoenzyme. (+info)
Analysis of the open region and of DNA-protein contacts of archaeal RNA polymerase transcription complexes during transition from initiation to elongation.
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The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation. We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation. RNAP was stalled between registers +5 and +20 using C-minus cassettes. The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position +5. Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register +8. A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers +6/+7. A major transition was observed at registers +10/+11. In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance. Between registers +11 and +20 both RNAP and transcription bubble moved synchronously with RNA synthesis. The distance of the catalytic center to the front edge of the exo III footprint was approximately 12 nucleotides in all registers. The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides. For complexes stalled between positions +10 and +20 the size of the transcription bubble was around 17 nucleotides. This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II. (+info)
On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter.
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Bacterial promoters of the extended -10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended -10 Escherichia coli galP1 promoter at temperatures as low as 6 degrees C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended -10 motif and/or containing the -35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif. However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent. Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif. We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations. (+info)
Kinetic spectrophotometric determination of famotidine in commercial dosage forms.
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A simple kinetic spectrophotometric method is described for the determination of famotidine. The method is based on the oxidation of the drug with alkaline potassium permanganate. The reaction is followed spectrometrically by measuring the rate of change of the absorbance at 610 nm. The initial-rate and fixed-time (at 12 min) methods are adopted for determining the drug concentration. The calibration graphs are linear in the ranges of 2-10 microg mL(-1) and 1-8 microg mL(-1) using the initial-rate and fixed-time methods, respectively. The method has been applied to the determination of famotidine in tablet formulations. The obtained results are compared statistically with those given by a reference spectrophotometric method. (+info)
Nucleotides from -16 to -12 determine specific promoter recognition by bacterial sigmaS-RNA polymerase.
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The alternative sigma factor sigmaS, mainly active in stationary phase of growth, recognizes in vitro a -10 promoter sequence almost identical to the one for the main sigma factor, sigma70, thus raising the problem of how specific promoter recognition by sigmaS-RNA polymerase (EsigmaS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EsigmaS at the strictly sigmaS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB -10 sequence (-12C), instead of the T nucleotide canonical for sigma70-dependent promoters, is the major determinant for selective recognition by EsigmaS. The presence of the -12C does not allow formation of an open complex fully proficient in transcription initiation by Esigma70. The role of -12C as specific determinant for promoter recognition by EsigmaS was confirmed by sequence analysis of known EsigmaS-dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that EsigmaS, unlike Esigma70, can recognize both C and T as the first nucleotide in the -10 sequence. Additional promoter features such as the presence of a C nucleotide at position -13, contributing to open complex formation by EsigmaS, and a TG motif found at the unusual -16/-15 location, possibly contributing to initial binding to the promoter, also represent important factors for sigmaS-dependent transcription. We propose a new sequence, TG(N)0-2CCATA(c/a)T, as consensus -10 sequence for promoters exclusively recognized by EsigmaS. (+info)
Flow-injection chemiluminescence determination of enalapril maleate in pharmaceuticals and biological fluids using tris(2,2'-bipyridyl)ruthenium(II).
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A chemiluminescence (CL) method using flow injection (FI) has been investigated for the rapid and sensitive determination of enalapril maleate. The method is based on the CL reaction of the drug with tris(2,2'-bipyridyl)ruthenium(II), Ru(bipy)3(2+) and acidic potassium permanganate. After selecting the best operating parameters, calibration graphs were obtained over concentration ranges of 0.005-0.2 microg/ml and 0.7-100 microg/ml with a detection limit (S/N=2) of 1.0 ng/ml. The average % found was 99.9 +/- 0.7 and 100.2 +/- 0.3 for the two concentration ranges respectively. %RSD (n=10) for 5.0 microg/ml was 0.44. The method was successfully applied to the determination of enalapril maleate in dosage forms and biological fluids without interferences. (+info)