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

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Abstract
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 complexs between RNA polymerase 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 correspondings to the lower template strand of lac UV5 in the region -25 to +10 relative to the transcriptions 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 of between -12 to +4 was position dependent, maximum reactivity being displayed by bases in the middle of the region. The reaction was first order with 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. The general shift in the reactivity profiles was taken as an indication that the primary determinant of the gradient of reactivity found in the single-stranded area was the position of the base. Our data challenge a too simplistic interpretation of most results obtained by chemical probing of nucleoprotein complexes. They suggest that, within unstacked and unwound regions of double-stranded DNA in open complexes, DMS and DEP reactions are limited by a base-specific structural reaggrangement.