lacP1 promoter with an extended -10 motif. Pleiotropic effects of cyclic AMP protein at different steps of transcription initiation.
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The cyclic AMP receptor protein (CRP), which activates transcription from the wild-type lacP1 promoter and most of its mutants, represses productive RNA synthesis from a lacP1 promoter variant that contains an extended -10 element, although CRP enhances RNA polymerase binding as well as open complex formation in both promoters. Moreover, abortive RNA synthesis, which is already higher in the extended -10 variant compared with the parent promoter, was further enhanced by CRP. These results, together with the observed decrease in productive RNA synthesis, indicate that CRP, while facilitating the earlier steps of initiation, inhibits transcription from the extended -10 lacP1 by hindering promoter clearance. We propose that CRP decreases energetic barriers to RNA polymerase binding, isomerization, and abortive RNA synthesis but stabilizes the abortive RNA initiating complex, which results in increasing the activation energy of the transition state before the elongation complex. The results demonstrate for the first time that a DNA-binding regulatory protein acts as an activator or a repressor in different steps of the transcription initiation pathway because of the energetic differences of the intermediate complex in the same promoter. (+info)
Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.
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We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions. (+info)
Requirements for DNA unpairing during displacement synthesis by HIV-1 reverse transcriptase.
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DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO(4) oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein. (+info)
Substitutions in region 2.4 of sigma70 allow recognition of the sigmaS-dependent aidB promoter.
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The strict dependence of transcription from the aidB promoter (PaidB) on the Esigma(S) form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the -10 promoter sequence (-12C), which does not allow an open complex formation by Esigma(70). In this report, sigma(70) mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow -12C recognition by sigma(70), were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.4 of sigma(70) and correspond to Gln-152 and Glu-155 in sigma(S). Interestingly, the Q437H mutant of sigma(70), but not T440I, was able to promote an open complex formation and to initiate transcription at PaidB. In contrast to T440I, a T440E mutant was proficient in carrying out transcription from PaidB. No sigma(70) mutant displayed significantly increased interaction with a PaidB mutant in which the -12C was substituted by a T (PaidB((C12T))), which is also efficiently recognized by wild type sigma(70). The effect of the T440E mutation suggests that the corresponding Glu-155 residue in sigma(S) might be involved in -12C recognition. However, substitution to alanine of the Glu-155 residue, as well as of Gln-152, in the sigma(S) protein did not significantly affect Esigma(S) interaction with PaidB. Our results reiterate the importance of the -12C residue for sigma(S)-specific promoter recognition and strongly suggest that interaction with the -10 sequence and open complex formation are carried out by different determinants in the two sigma factors. (+info)
TFIIH operates through an expanded proximal promoter to fine-tune c-myc expression.
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A continuous stream of activating and repressing signals is processed by the transcription complex paused at the promoter of the c-myc proto-oncogene. The general transcription factor IIH (TFIIH) is held at promoters prior to promoter escape and so is well situated to channel the input of activators and repressors to modulate c-myc expression. We have compared cells expressing only a mutated p89 (xeroderma pigmentosum complementation group B [XPB]), the largest TFIIH subunit, with the same cells functionally complemented with the wild-type protein (XPB/wt-p89). Here, we show structural, compositional, and functional differences in transcription complexes between XPB and XPB/wt-89 cells at the native c-myc promoter. Remarkably, although the mean levels of c-Myc are only modestly elevated in XPB compared to those in XPB/wt-p89 cells, the range of expression and the cell-to-cell variation of c-Myc are markedly increased. Our modeling indicates that the data can be explained if TFIIH integrates inputs from multiple signals, regulating transcription at multiple kinetically equivalent steps between initiation and promoter escape. This helps to suppress the intrinsic noise of transcription and to ensure the steady transcriptional output of c-myc necessary for cellular homeostasis. (+info)
Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases.
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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. (+info)
Flow injection chemiluminescence analysis of some penicillins by their sensitizing effect on the potassium permanganate-glyoxal reaction.
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A new chemiluminescence method using flow injection is described for the determination of four penicillins, namely: phenoxymethylpenicillin potassium, amoxicillin, ampicillin, and ampicillin sodium. The method is based on sensitizing effect of these drugs on the chemiluminescence reaction of potassium permanganate in sulfuric acid with glyoxal. The different experimental parameters affecting the chemiluminescence intensity were carefully studied and incorporated into the procedure. The method allows the determination of 0.1-1.0 microg/ml phenoxymethylpenicillin potassium, 0.1-1.0 microg/ml amoxicillin, 0.1-1.0 microg/ml ampicillin, and 0.1-1.0 microg/ml ampicillin sodium. The method was successfully applied to the determination of four penicillin antibiotics in pharmaceutical preparations. (+info)
Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage lambda Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo. (+info)