title: Influence of Mg2+ and temperature on formation of the transcription bubble.
abstract: The transcription bubble formed in the binding complex of T7A1 promoter upon Escherichia coli RNA polymerase was analyzed by chemical probes, namely by single-strand specific reagents, to map the unpaired bases in the bubble, and by FeEDTA, to analyze the accessibility of the DNA backbone. The latter probe could also be used as a local hydroxyl radical probe placed close to the Mg2+-binding site in the active center. The data show that the transcription bubble consists of two parts, an Mg2+-dependent part and an Mg2+-independent part, both having individual transition temperatures. The data further suggest that formation of a transcription active open complex is preceded by a transition state complex having enhanced affinity for those Mg2+ ions presumably participating in the formation of the catalytic site. Our data also suggests that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent. One/two of the three Mg2+ ions are responsible for the polymerization, the other two/one for enlargement of the transcription bubble.
authors.affiliation: Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Zaychikov
authors.forename: L
authors.initials: L
authors.lastname: Denissova
authors.forename: T
authors.initials: T
authors.lastname: Meier
authors.forename: M
authors.initials: M
authors.lastname: Götte
authors.forename: H
authors.initials: H
authors.lastname: Heumann
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004214:Distamycins / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D017665:Hydroxyl Radical; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D004214:Distamycins; D009942:Organometallic Compounds; C038558:distamycin-EDTA-iron(II); D015636:Magnesium Chloride; D017665:Hydroxyl Radical; D004492:Edetic Acid; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium