Fine mapping of DNA single-stranded regions using base-specific chemical probes: study of an open complex formed between RNA polymerase and the lac UV5 promoter.
We have used diethyl pyrocarbonate (DEP), which carbethoxylates adenine bases, and dimethyl sulfate (DMS), which methylates guanine residues and single-stranded cytosines, to probe bases in open complexes between RNA polymerase and the lac UV5 promoter in vitro. We compared the kinetics of reactivity between bases in an open complex and those in a single-stranded 35-mer fragment corresponding to the lower template strand of lac UV5 in the region -25 to +10 relative to the transcription start site. We observed that cytosine and adenine residues in the 35-mer fragment reacted according to a second-order process with DMS and DEP, respectively, at sufficiently low concentrations of the reagents and that the degree of reactivity was base position independent. In an open complex in the absence of substrates, we observed reactivity with DEP in adenines from -12 to +4 as well as +21 on the template strand and methylation by dimethyl sulfate of cytosines -6, -4, -2, and -1. No hyperreactivity was observed on the nontemplate strand. The degree of reactivity of bases between -12 and +4 was position dependent, maximum reactivity being displayed by bases in the middle of the region. The reaction was first order within the range of reagent concentration investigated. It was confirmed that in the presence of ApA and UTP cytosine +5, as well as cytosines -6, -4, -2, and -1, in an open complex became reactive to DMS. With regard to DEP the extent of reactivity of the adenine at position +3 was increased markedly, adenine +4 was brought into the single-stranded region, and the overall reactivity of adenine -10 decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
Buckle
M
M
Unité de Physicochimie des Macromolécules Biologiques, Institut Pasteur, Paris, France.
Buc
H
H
D000225:Adenine; D002874:Chromosome Mapping; D003596:Cytosine; D004277:DNA, Single-Stranded / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004047:Diethyl Pyrocarbonate; D015335:Molecular Probes; D011401:Promoter Regions, Genetic; D013463:Sulfuric Acid Esters
D004277:DNA, Single-Stranded; D015335:Molecular Probes; D013463:Sulfuric Acid Esters; D003596:Cytosine; D012321:DNA-Directed RNA Polymerases; D000225:Adenine; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate