Effect of supercoiling on the abortive initiation kinetics of the RNA-I promoter of ColE1 plasmid DNA.

D C Wood,J Lebowitz

doi:10.1016/s0021-9258(18)90844-6

Abstract

An analysis of the effect of DNA supercoiling on transcription of the RNA-I promoter of ColE1 has been undertaken using the abortive initiation assay developed by McClure (McClure, W. R., Cech, C. L., and Johnston, D. E. (1978) J. Biol. Chem. 253, 8941-8948). A tau plot was constructed, enabling the equilibrium constant for closed complex formation (KI) and the isomerization rate constant (k2) to be determined. The effect of supercoiling on RNA-I is an apparent 100-fold increase in the isomerization rate, k2, with little or no effect on KI. This is the first reported abortive initiation analysis of RNA-I transcription which serves in the regulation of plasmid replication.

Highlights

This approach allows a quantitative analysis of the strength of a promoter and shows that supercoiling produces a 100-fold increase inthe rate of isomerization from a closed to an open promoter-polymerase complex without affecting the binding constant for the closed state
The effect of supercoiling on RNA-I is an apparent 100-fold increase in the isomerization rate, k2,with little or no effect on K I. This is the first reported abortive initiation analysis of RNA-I transcription which serves in the regulation of plasmid replication
Preparation of ColEl DNA-ColE1 plasmid was prepared from an E. coli K12 strain [17] as previously described [18].DNA prepared in this manner was shown to be greater than 90% form I by agarose gel electrophoresis

Summary

Introduction

This approach allows a quantitative analysis of the strength of a promoter and shows that supercoiling produces a 100-fold increase inthe rate of isomerization from a closed to an open promoter-polymerase complex without affecting the binding constant for the closed state. An analysis of the effect of DNA supercoiling on transcription of the RNA-I promoter of ColEl has been undertaken using the abortive initiation assay developedbyMcClure(McClure, W.

Results

Conclusion