SignificanceMany bacteria produce antimicrobial peptides for survival under stressful conditions. Some of these antimicrobial peptides are lasso peptides, which have a unique lasso-like topology and have generated great interest as a result of their stability in harsh conditions and amenability to functional engineering. In this study, we determined crystal structures of two lasso peptides, microcin J25 and capistruin, bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). The structures define peptide inhibitor–RNAP interactions that are important for inhibition and provide detailed insight into how the peptides inhibit RNAP function. This work provides a structural basis to guide the design of more potent lasso peptide antimicrobial approaches.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.

The 5 0 -nucleotide of small RNAs associates directly with the MID domain of Argonaute (AGO) proteins. In humans, the identity of the 5 0 -base is sensed by the MID domain nucleotide specificity loop and regulates the integrity of miRNAs. In Arabidopsis thaliana, the 5 0 -nucleotide also controls sorting of small RNAs into the appropriate member of the AGO family; however, the structural basis for this mechanism is unknown. Here, we present crystal structures of the MID domain from three Arabidopsis AGOs, AtAGO1, AtAGO2 and AtAGO5, and characterize their interactions with nucleoside monophosphates (NMPs). In AtAGOs, the nucleotide specificity loop also senses the identity of the 5 0 -nucleotide but uses more diverse modes of recognition owing to the greater complexity of small RNAs found in plants. Binding analyses of these interactions reveal a strong correlation between their affinities and evolutionary conservation.

SUMMARY Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and sub-unit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.