Nucleoside Triphosphate Requirement for Termination of RNA Synthesis by Rho Factor

Abstract

INTRODUCTION
Rho factor, a protein isolated from Escherichia coli, causes termination of RNA synthesis catalyzed in vitro by RNA polymerase at distinct sites on DNA templates (Roberts 1969). Rho also catalyzes an RNA-dependent hydrolysis of nucleoside triphosphates to nucleoside diphosphates and orthophosphate (Lowery-Goldhammer and Richardson 1974). Although this hydrolysis reaction can occur in complete RNA synthesis reaction mixtures where rho is acting as a termination factor, there is no obvious functional relationship between the two activities; isolated RNA molecules are alone sufficient to activate the rho-catalyzed hydrolysis reaction, and under the conditions where rho does cause termination, the ATP hydrolysis reaction continues long after the termination event, so that by 90 minutes, the number of ATP molecules hydrolyzed exceeds the number of chains terminated by more than a thousand to one. However in spite of this apparent lack of coupling, the rho-mediated termination reaction could be dependent on a reaction with a nucleoside triphosphate. This paper presents evidence that there is a dependence.

Two experiments are presented. One experiment shows that the rho ATP hydrolysis reaction in complete RNA polymerase reaction mixtures has a sensitivity to salt similar to that of the termination activity. The other experiment shows that rho is unable to function as a termination factor when the nucleoside triphosphates are replaced by analogs that are substrates for the RNA polymerase-catalyzed reaction but not for the nucleoside triphosphate hydrolysis reaction.

MATERIALS
RNA polymerase was purified from E. coli B as described previously (Richardson 1973). Rho factor was purified...