Abstract
Addition of 10 to 100 oligodeoxycytidylate residues to the 3'-OH termini of T7 bacteriophage DNA produces a highly efficient template for transcription in vitro with purified calf thymus RNA polymerase II. Transcription initiates rapidly and selectively at the oligo (dC) ends of such a template and essentially all of the active RNA polymerase II molecules are then committed to a long period of RNA chain elongation. This allows the direct study of the RNA chain elongation and termination reactions and also permits determination of the concentration of active RNA polymerase II that is present. From 15 to 25% of the RNA polymerase molecules in our current preparations are active in these reactions. RNA chain elongation by calf thymus RNA polymerase II is relatively slow (7 nucleotides/s) even at saturating substrate concentrations. The in vitro elongation process appears to be discontinuous, with elongating polymerase molecules pausing for significant periods at certain sequences along the DNA. There is a low, but measurable frequency of RNA chain termination at some sites; however, the majority of elongating transcripts can grow to large sizes (over 6000 nucleotides). Surprisingly, over 60% of the active calf thymus RNA polymerase II molecules form a long DNA-RNA hybrid during in vitro transcription and displace the nontranscribed DNA from the template to produce a characteristic split end structure. DNA-RNA hybrids are also formed during transcription by RNA polymerase II from duplex DNA templates lacking 3' oligo(dC) tails, which takes place predominantly at single strand breaks or ends. Thus the transcriptional elongation reaction carried out by calf thymus RNA polymerase II in vitro differs in several respects from that which must take place in vivo.
Highlights
Addition of 10 t o 100 oligodeoxycytidylate residues the different steps in the transcription cycle and to study the to the3”OH termini of T7 bacteriophage DNA produces elementary steps in the reactions involved
Rationale-Under in vitro conditions where a population of RNA polymerase molecules initiates transcription rapidly, and continues RNA chain elongation for a substantial time without termination and repeated initiation, the amount of catalytically active RNA polymerase can be determined directly from the rateof nucleotide incorporation [6].Such an experiment can givevalues for many of the basic parameters that characterize the individual steps of the transcription cycle
Current preparations of RNA polymerase I1 do not carry out accurate transcription of eukaryotic or viral genes on duplex DNA [1, 2] and while factors have recently been identified that stimulate accurate transcription [3,4,5], these systems are still very inefficient whencompared to transcription in intact nuclei
Summary
Escherichia coli RNA polymerase holoenzyme was purified by the out by RNA polymerase I1we chose to bypass the need for method of Gonzalez ef al. [12] andhada specific activity of220 actual promoter sites for polymerase 11, which are still used milliunits/mg of protein where 1 unitcatalyzes a rate of incorporation 1 q o l of CMP/min with T7 DNA as template under the assay conditions of Chamberlin et at. ( 6 ) .E . coli core RNA polymerase was purified by poly(rC)-cellulose chromatography using an unpublished procedure of R. Kingston and was totally free of contaminating u veryinefficientlyin current in vitro systems It has been known for some time that purified eukaryotic RNA polymerase I1 preparations have poor activity with intact duplex templates, but preferentially bind to, and initiate chains at subunit as judged by both transcriptional analysis with T7 DNA and single strand breaks or in single-stranded regions of the tempeptide analysis by SDS-polyacrylamide gels. Specificity of Transcription by Calf Thymus RNA Polymerase II Using p C T 7 DNA Templates"T7 DNA to which tails of approximately 50-75 nucleotides in length. This DNA was purified by phenol extraction followed by dialysis into asolution containing 10 mM Tris-C1pH 8.0, 50 m~ NaCl, and 0.1 m~ EDTA. Dried gels were autoradiographed at -70 "C using c40 an intensifying screen
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