Effect of chemical modification of supercoiled simian virus 40 DNA on the rate of in vitro transcription.

Superhelical simian virus 40 FI DNA could be modified with the single-strand-specific reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC). A limited reaction, of less than 2% of the base pairs, resulted in almost total inhibition of in vitro transcription by DNA-dependent RNA polymerase from Escherichia coli. This effect was shown to be due to DNA modification and not to inhibition of polymerase activity by the reagent. Inhibition of enzyme activity occurred if the contaminating reagent was not absorbed with another protein before polymerase addition. No inhibition was observed when DNA and polymerase were incubated together to allow the formation of pre-initiation complexes before CMC was added. Studies of template saturation with polymerase showed that the inhibition of transcription by DNA modification was due to a loss of binding ability of the enzyme to the reacted, supercoiled DNA when reaction times of less than 2 h were used.