Studies on transcription of 3'-extended templates by mammalian RNA polymerase II. Parameters that affect the initiation and elongation reactions

Abstract

Addition of short sequences of dCMP residues to the 3'-OH end of duplex linear DNAs allows rapid and efficient transcription to be initiated at these sites by purified mammalian RNA polymerase II [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. The use of such tailed DNA templates should allow biochemical studies on transcription elongation and termination with almost any desired DNA sequence. However, in vitro transcription with RNA polymerase II is aberrant in that the DNA template is not re-formed after transcription; rather, the DNA strands are separated, and most of the RNA product is found as a DNA-RNA hybrid. To better understand the factors that affect the process of transcription with these tailed DNA templates, we have varied a number of parameters that might be expected to play a role in the reaction. RNA polymerase II preparations from calf thymus, HeLa cells, and Drosophila all fail to displace the product RNA. However, RNA polymerase II from wheat germ gives only free RNA as a product, as does the Escherichia coli RNA polymerase. Hence, the displacement of the nascent RNA from a transcription complex seems to depend on some intrinsic property of the polymerase itself and not simply on the nature of the template. Variation of reaction conditions, or of the divalent metal ion, does not restore the renaturability of the DNA template. However, variation of the duplex 3'-terminal sequence of the template led to significant alterations. In general, GC-rich sites enhanced the displacement of the nascent RNA, while AT-rich sites enhanced formation of the DNA-RNA hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)