INFLUENCE OF MG2+ AND TEMPERATURE ON FORMATION OF THE TRANSCRIPTION BUBBLE

The transcription bubble formed in the binding complex of T7A1 promote r 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 rad ical 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 individ ual transition temperatures, The data further suggest that formation o f a transcription active open complex is preceded by a transition stat e complex having enhanced affinity for those Mg2+ ions presumably part icipating in the formation of the catalytic site, Our data also sugges ts that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent, One/two of the three Mg2+ ions are respo nsible for the polymerization, the other two/one for enlargement of th e transcription bubble.