FIGURE 8.

Model accounting for the action of CTD tail on E. coli gyrase. Arrow thickness indicates the qualitative probability of transitioning between states. Panel i, full-length GyrA cannot wrap DNA on account of the repressive action of the tail on CTD activity and/or localization. Panels ii and iii, binding of GyrB releases the repressive activity, either allosterically or by binding the tail directly, allowing wrapping. Panel iv, ATP binding induces strand passage, freeing the tail from the action of GyrB; hydrolysis allows reset back to state (panel iii). Successive rounds of turnover allow for DNA supercoiling, which is extensive due to the tight coupling of CTD wrapping with ATPase status. Panel v, futile cycle in which ATP binding is accidentally not converted to a strand passage event, which is possibly due to incomplete wrapping. In wild-type gyrase, this event is rare (38, 45), but it may become more prevalent as DNA becomes more negatively supercoiled. Panel vi, AMP-PNP permanently relieves the effect of GyrB on the tail, allowing re-engagement of the CTD to prohibit wrapping. Note that our data neither support nor exclude the possibility that a similar, tail-repressed CTD state might occur during normal enzyme cycling. Panel vii, removal of the tail frees the CTD to wrap DNA in the context of GyrA alone. Panel viii, GyrB does not potentiate, and may even hinder, wrapping in the context of the holoenzyme. Panel ix, futile cycles of ATPase activity occur with increased frequency on account of the loss of coupling between CTD activity and GyrB status. Panel x, occasional ATPase cycles result in strand passage. The degree of supercoiling obtained without the tail is not as extensive as in wild-type gyrase, either because the inefficiency of the enzyme permits a relaxation “back-reaction” to occur (i.e. tail-less gyrase acts like a nonsupercoiling type II topoisomerase) or because the ability of the enzyme to maintain a wrapped DNA state cannot compensate as its “load” is increased (i.e. the wrap on the CTD slips as DNA becomes more supercoiled). At present, we cannot distinguish between these two possibilities. Panel xi, AMP-PNP has no effect on wrapping by a tail-less enzyme because the CTD is no longer under the control of GyrB. NTD, N-terminal domain.