Steady-state kinetic analysis of transcription of cloned tRNASer genes from Drosophila melanogaster

Abstract

Drosophila melanogaster Schneider II cells contain a factor which inhibits transcription in vitro of cloned tRNA genes in crude extracts made from these cells. The inhibitor could, however, be effectively neutralized by addition of certain non-template DNAs. In the absence of the transcription inhibitor activity, the steady-state kinetics of tRNA production from cloned genes followed one-substrate enzyme kinetics to a high degree of accuracy. Maximal rates of transcription and apparent affinity constants were analyzed for a collection of cloned D. melanogaster tRNASer genes. The stability of the complex formed by the transcription proteins and the template DNA was found to be nearly constant for the genes examined. The transcription rates, however, were greatly influenced by the DNA sequences flanking the tRNA genes. Analysis of transcription competition between DNA templates showed pure competitive behavior. Inhibition constants derived from these experiments indicated that the formation of the transcription complex was affected by sequences flanking the tRNA genes. Furthermore, the rate-limiting step in complex formation was independent of the stability of the final form of the complex.