Inhibition of transcription of supercoiled PM2 DNA by carbodiimide modification

Abstract

PM2 superhelican DNA (form I), which as been reacted with the single strand specific reagent, N-cyclohexyl-N'-beta-(methylmorpholinium)ethyl carbodiimide (CMC) is more than 95% inhibited in its ability to support transcription with E. coli B RNA polymerase in vitro. Almost complete inhibition of transcription was achieved after 2 hours of reaction with FI when only 1% of the bases were modified. A large increase in S20,* (from 26.8 S to 33.6 S) of FI DNA was observed during the course of reaction. Rifampicin resistant transcription is more susceptible to inhibition by CMC than total transcription, suggesting that the CMC is preferentially binding at promoter sites. These results clearly are in accord with the observation that supercoiled DNA contains localized regions of unpaired bases. The promotor sites for E. coli RNA polymerase in FI PM2 DNA appear to be located at or near these unpaired sites.

Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli

“…Little difference was observed between FI with rifampin added with the triphosphates and FI with no rifampin added. These results are in agreement with those of Mandel and Chambon (13) for SV40 and those obtained in the 4X174, PM2, and A systems, where, respectively, 20-, 10-, and 5-fold differences were observed for transcription of Fl DNA over FII DNA in the presence of rifampin (1,5,8,16).…”

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

“…This single-strand specific reagent has served as a probe for unpaired and mismatched sites in DNA [3,4]. CMC-reacted DNA has also been shown to be inhibited in its ability to support transcription by RNA polymerase [5]. One of the most powerful applications of CMC derivatization has been its use as a sequencing tool for the detection of the posttranscriptionally modified nucleoside, pseudouridine (Ψ), in RNA [6][7][8][9][10].…”

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

“…Single-stranded regions of SV40 DNA could arise from natural protein-DNA interactions as well as from supercoiling and may be involved in both in vivo as well as the in vitro transcription that we have studied here. Inhibition of in vitro transcription by CMC reaction has also been observed with PM2 DNA (6). Also, a recent electron microscopic comparison of transcription on linear and superhelical bacteriophage A DNA (2) has shown an increased activity of existing promoters and the production of new initiation sites at adeninethymine-rich, easily denatured, regions on supercoiled DNA.…”

“…Supercoiled (FI) DNA has been shown in several systems (2,3,6, 10, 18,19,22) and in simian virus 40 (SV40) (15; see accompanying paper [9]) to act as a more efficient template for RNA synthesis by DNA-dependent RNA polymerase than relaxed, or nonsupercoiled (FIl), DNA. Unpaired regions in supercoiled DNA have been indicated by a number of different methods (12; 21 reviews literature through 1976).…”