A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a β-roll upon binding the -paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding -paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the β3/β4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest N backbone chemical shift differences are associated with the β3/β4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.