NUCLEASE ACTIVITY OF T7 RNA-POLYMERASE AND THE HETEROGENEITY OF TRANSCRIPTION ELONGATION COMPLEXES

We have discovered that T7 RNA polymerase, purified to apparent homoge neity from overexpressing Escherichia coli cells, possesses a DNase an d an RNase activity, Mutations in the active center of T7 RNA polymera se abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs, Under the conditions o f promoter-driven transcription on double-stranded DNA, no nuclease ac tivity was observed, The nuclease attacks DNA oligonucleotides in mono - or dinucleotide steps, The nuclease is a 3' to 5' exonuclease leavin g a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+, The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely, In the presence of rNTPs, the nuclease activity is sup pressed but an unusual 3'-end-initiated polymerase activity is unmaske d, RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligon ucleotide template exhibited evidence of nuclease activity. The nuclea se activity of T7 RNA polymerase is unrelated to pyrophosphorolysis, W e propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unus ual 3'-end polymerizing activity, causes heterogeneity in elongation c omplexes, Additionally, during normal transcription elongation, the ki netic balance between nuclease and polymerase is shifted in favor of p olymerase.