Inactivation of purified Escherichia coli RNA polymerase by transfer RNA

Abstract

The inhibition by transfer RNA of the in vitro activity of purified RNA polymerase isolated from Escherichia coli has been investigated. Reaction of polymerase with tRNA§ before the addition of DNA to the reaction mixture inhibits the synthetic activity of the polymerase to an extent which depends upon the relative concentrations of tRNA and polymerase. Prior reaction of the polymerase with DNA protects it against subsequent inactivation by tRNA. Thus reaction of the polymerase with either nucleic acid appears to be practically irreversible. The inhibitory effect of tRNA on a polymerase molecule is all-or-none, since partial inhibition of RNA synthesis by tRNA reduces only the number of growing RNA chains, but not their rate of growth. The nature of the dependence of relative inhibition on tRNA concentration suggests that the populations of polymerase and tRNA molecules are heterogeneous with respect to their mutual affinities. This possibility is supported by the finding that in a mixture of polymerase and tRNA at concentrations such that half of the polymerase activity is inhibited, there still exists tRNA capable of inhibiting freshly added polymerase. It was found that aminoacyl-tRNA is a somewhat less effective inhibitor than amino acid-free tRNA, although this difference does not appear to be great enough to account for the postulated in vivo regulation of RNA synthesis by tRNA.