Transcription initiation at the flagellin promoter by RNA polymerase carrying sigma28 from Salmonella typhimurium.
The sigma subunit of RNA polymerase is a critical factor in positive control of transcription initiation. Primary sigma factors are essential proteins required for vegetative growth, whereas alternative sigma factors mediate transcription in response to various stimuli. Late gene expression during flagellum biosynthesis in Salmonella typhimurium is dependent upon an alternative sigma factor, sigma28, the product of the fliA gene. We have characterized the intermediate complexes formed by sigma28 holoenzyme on the pathway to open complex formation. Interactions with the promoter for the flagellin gene fliC were analyzed using DNase I and KMnO4 footprinting over a range of temperatures. We propose a model in which closed complexes are established in the upstream region of the promoter, including the -35 element, but with little significant contact in the -10 element or downstream regions of the promoter. An isomerization event extends the DNA contacts into the -10 element and the start site, with loss of the most distal upstream contacts accompanied by DNA melting to form open complexes. Melting occurs efficiently even at 16 degrees C. Once open complexes have formed, they are unstable to heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at rRNA promoters. (+info
Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol.
Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA hetero-duplexes. Potassium permanganate in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues. The DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches. KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without disrupting the sensitivity and efficiency of either reaction. (+info
Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC.
Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC. (+info
Strand opening by the UvrA(2)B complex allows dynamic recognition of DNA damage.
Repair proteins alter the local DNA structure during nucleotide excision repair (NER). However, the precise role of DNA melting remains unknown. A series of DNA substrates containing a unique site-specific BPDE-guanine adduct in a region of non-complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA. UvrBC formed a pre-incision intermediate with a DNA substrate containing a 6-base bubble structure with 2 unpaired bases 5' and 3 unpaired bases 3' to the adduct. Formation of this bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base. The dual incisions were strongly determined by the distance between the adduct and the double-stranded-single-stranded DNA junction of the bubble, and required homologous double-stranded DNA at both incision sites. Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of >/=>/=10 nucleotides. These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner. (+info
Simple method for determination of thiocyanate in urine.
BACKGROUND: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations. METHODS: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0-100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 x absorbance. RESULTS: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r(2) = 1. 000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method. CONCLUSIONS: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries. (+info
Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating.
Gating of the cystic fibrosis Cl(-) channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein. With Ca(2+) as the dominant divalent cation instead of Mg(2+), the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca(2+). In contrast, "soft" divalent cations such as Cd(2+) interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity. (+info
Mechanism of repression of the aroP P2 promoter by the TyrR protein of Escherichia coli.
Previously, we have shown that expression of the Escherichia coli aroP P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the aroP P2 promoter. In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes. We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression. The TyrR binding site of aroP, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2. Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression. (+info
TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.
TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients. (+info