Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes.
We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase 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 of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving 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 suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.
Sastry
S S
SS
Laboratory of Molecular Genetics, Box 174, The Rockefeller University, New York, New York 10021, USA.
Ross
B M
BM
D017123:Bacteriophage T7; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D011564:Furocoumarins / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism; D010445:Peptide Elongation Factors; D012313:RNA / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D011564:Furocoumarins; D010445:Peptide Elongation Factors; D014764:Viral Proteins; D012313:RNA; D006493:Heparin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases; D012260:Ribonucleases; D009238:N-Acetylmuramoyl-L-alanine Amidase; D008274:Magnesium