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It was shown previously, that at moderate excess of RNA polymerase (RNAP) relative to DNA, the complex of the bla promoter from bacterial transposon Tn3 with RNAP is locked in an inactive, heparin-resistant, isomeric state which is not that of the 'open' complex. This 1:1 isomer can accommodate a second RNAP, which becomes tightly and specifically bound just upstream of the first RNAP [Duval-Valentin &amp; Reiss (1990) Mol. Microbiol. 4, 1465-1475]. Both the resulting 2:1 complex and its antecedent 1:1 complex formed at excess of RNAP are immediately and permanently inhibited for transcription initiation. Using the gel-retardation technique, we investigate here the kinetics of formation and decay of the 2:1 complex under various experimental conditions. The data are consistent with pseudo-first-order kinetics at moderate excess of RNAP. The salt-dependence of rate and equilibrium constants has been analysed within the framework of the theoretical model described by Lohman, Dehaseth &amp; Record [(1978) Biophys. Chem. 8, 281-294]. It was found that the salt-dependence is consistent with the existence of a transient intermediate during formation of the 2:1 complex, which forms rapidly on the time scale of its isomerization to the final 2:1 complex. The intermediate is characterized by the release of about seven cations from the 1:1 complex, one additional cation being released upon its final isomerization. Formation of the 2:1 complex at high excess of RNAP becomes inhibited, probably as a result of a 'bumping' effect of the complex by the enzyme, also observed with several other promoters. We conclude that formation of the 2:1 complex closely mimics that of the standard 1:1 complex, except that the final isomerization step to an 'open' complex is lacking. A mechanism of the formation of the 2:1 complex and of its role in transcription regulation of constitutive promoter by RNAP is proposed.

Kinetics of the specific binding of a second RNA polymerase to the standard bacterial-transposon-Tn3 bla promoter complex