Evidence for a compact σ70 conformation in vitro and in vivo

Highlights•smFRET and CL-MS point toward a compact conformation of apo-σ70•Structural basis for inhibition of σ70's DNA binding when not part of transcription•Apo-σ70 in its compact conformation exists in vivo during stationary phaseSummaryThe initiation of transcription in Escherichia coli (E. coli) is facilitated by promoter specificity factors, also known as σ factors, which may bind a promoter only as part of a complex with RNA polymerase (RNAP). By performing in vitro cross-linking mass spectrometry (CL-MS) of apo-σ70, we reveal structural features suggesting a compact conformation compared to the known RNAP-bound extended conformation. Then, we validate the existence of the compact conformation using in vivo CL-MS by identifying cross-links similar to those found in vitro, which deviate from the extended conformation only during the stationary phase of bacterial growth. Conclusively, we provide information in support of a compact conformation of apo-σ70 that exists in live cells, which might represent a transcriptionally inactive form that can be activated upon binding to RNAP.Graphical abstract