Concerted transformation of a hyper-paused transcription complex and its reinforcing protein.

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a ß-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.

Exploring the structural acrobatics of fold-switching proteins using simplified structure-based models.

Metamorphic proteins are a paradigm of the protein folding process, by encoding two or more native states, highly dissimilar in terms of their secondary, tertiary, and even quaternary structure, on a single amino acid sequence. Moreover, these proteins structurally interconvert between these native states in a reversible manner at biologically relevant timescales as a result of different environmental cues. The large-scale rearrangements experienced by these proteins, and their sometimes high mass interacting partners that trigger their metamorphosis, makes the computational and experimental study of their structural interconversion challenging. Here, we present our efforts in studying the refolding landscapes of two quintessential metamorphic proteins, RfaH and KaiB, using simplified dual-basin structure-based models (SBMs), rigorously footed on the energy landscape theory of protein folding and the principle of minimal frustration. By using coarse-grained models in which the native contacts and bonded interactions extracted from the available experimental structures of the two native states of RfaH and KaiB are merged into a single Hamiltonian, dual-basin SBM models can be generated and savvily calibrated to explore their fold-switch in a reversible manner in molecular dynamics simulations. We also describe how some of the insights offered by these simulations have driven the design of experiments and the validation of the conformational ensembles and refolding routes observed using this simple and computationally efficient models.

DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility.

Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.