Bacteriophage T7 RNA polymerase. 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript.
We have substituted 5-fluorodeoxyuridine (5-FdU) in place of thymidine in defined positions along synthetic bacteriophage T7 promoter DNA sequences. None of the fluoro-substitutions in the promoter DNA sequence reduced transcription yields with T7 RNA polymerase significantly. Substitutions on the coding template strand reduced transcription yields when placed at +3, but not at +4. 19F-n.m.r. spectra from transcription reactions and gel analysis of transcription products show that T7 RNA polymerase correctly and efficiently utilizes 5-FUTP as a RNA substrate analog. The fluorine atom provides a sensitive probe for monitoring the local environment, base sequence and solvent exposure at the DNA major groove through its 19F-n.m.r. resonance. Buffer dependencies of the fluorine chemical shift and digestion patterns with DNase I suggest that the T7 promoter base-pairs near the transcription start site are distorted with a more open minor groove and less solvent accessible major groove. Previous chemical footprinting data of promoter-polymerase complexes yield a picture that T7 RNA polymerase recognizes major groove features in the region from positions -7 to -11 and minor groove features on the same side of DNA flanking both sides of this region. Consistent with this, 19F-n.m.r. observations identify two additional positions, -8 and -17, involved in promoter recognition on this side of the DNA helix. On the other hand, our observations also implicate the opposite side of the DNA helix, primarily at positions -14 and -15, as major groove recognition sites for T7 RNA polymerase. In addition, n.m.r. spectra from 5-FdU-substituted base-pairs -2 and -3, suggest either additional interactions on the same side of the DNA helix as -14 and -15, or distortions in the DNA structure.
Rastinejad
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Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Lu
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D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D005472:Fluorouracil / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D005472:Fluorouracil