Structural dynamics underlying the multi-functionality of a global gene regulator

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More than 90 % of all bacterial species with sequenced genomes express the hexameric RNA-dependent NTPase, p. p is essential in many of these bacteria. p is long known as a mediator of transcription termination that defines, for instance, the ends of 20-30 % of transcription units in Escherichia coli. However, recent findings have uncovered an unexpected multi-functionality of p, through which p acts as a global gene regulator and as a phenotypic heterogeneity factor that increases chances for survival of bacteria under adverse conditions. For example, p can, among others, also mediate attenuation in 5’-untranslated regions, limit the extent of antisense transcription, silence foreign genes and safeguard genomes by restricting R-loops. The multi-functionality of p is apparently established through its direct or indirect interactions with other proteins and RNA elements. Despite decades of research, the molecular mechanisms underlying ρ-dependent transcription termination and how p-interacting proteins and RNAs modulate and diversify p activity and function are poorly understood. This project will provide a comprehensive view on the structural basis of ρ’s dynamic interactions with transcription elongation complexes as well as with other proteins and RNAs that modulate ρ activity on elongation complexes, and will reveal how these interactions facilitate p’s many roles in cells. It involves capturing and structurally analyzing intermediate stages and regulatory situations along NTP-driven, multi-step processes that are mediated by large and flexible molecular machinery, and concomitantly delineating the conformational and compositional changes that these macromolecular complexes undergo during these processes. Moreover, structural insights will be exploited to guide functional analyses in vitro and in vivo, including genome/transcriptome-wide investigations. Assembling carefully designed, p-modified and p-modulating ECs and capitalizing on recent breakthroughs in single-particle cryo-electron microscopy, we have already made major progress towards these ambitious goals. Expected results will uncover fundamental principles of bacterial gene regulation, and methods developed will provide a blueprint for the stage-specific analysis of dynamic macromolecular complexes, to obtain movie-like representations in atomic detail of reaction cycles beyond the specific systems studied here.