IN VITRO TRANSCRIPTION OF Ad2 DNA BY EUKARYOTIC RNA POLYMERASES II. II. TRANSCRIPTION FROM STABLE BINARY COMPLEXES

Abstract

This chapter discusses the in vitro transcription of Ad2 DNA by eukaryotic RNA polymerase II. The mechanism of initiation of transcription from stable binary complexes between eukaryotic RNA polymerase II has been studied. RNA polymerase binds stably to DNA, forming I. I complexes are converted into MRSM. In the presence of ribonucleotide triphosphates, each RS complex can catalyze the rapid formation of the first phosphodiester bond in the nascent RNA chains. M1 of 9 mapped stable binary complexes are able to initiate RNA chains. The transition from I to RS complex exhibits a sigmoidal dependence on temperature for which a melting temperature ( T m ) can be determined. The T m for these polymerases is 22–25°C. A plot of the temperature dependence suggests the opening of 4–8 nucleotide base pairs on Ad2 DNA by these polymerases. This is the same as the number of base pairs opened by E. coli RNA polymerase. The direction of synthesis from the stable binding sites has been determined by two independent methods which include electron microscopic mapping of the position of DNA:RNA polymerase:RNA tertiary complexes as a function of time of RNA chain synthesis and hybridization of synthesized RNA to DNA blots of separated strands of various Ad2 DNA fragments.