Sequence-specific RNA-binding proteins (RBPs) must efficiently locate their targets among a multitude of cellular RNAs. Cas13, an RNA-guided CRISPR protein, represents an ideal model system in which to study this search process. Cas13 combats bacteriophage infection by cleaving RNA nonspecifically upon binding of its crRNA to the target RNA sequence; thus, Cas13’s search for its RNA target comes with a time constraint determined by the rate of phage multiplication. The mechanism by which Cas13 locates its target within this critical window remains unknown. Here, we investigate Cas13’s mechanism of target search through integration of biophysical modeling, activity assays, and biochemical characterization. We show that Cas13 employs facilitated diffusion to accelerate its search, and find that Cas13’s search time when targeting RNAs of different lengths cannot be explained by 1D sliding, the search mechanism used by many DNA-binding proteins. We propose that Cas13 primarily searches for its RNA target by intersegmental transfers (ITs), non-specifically binding the RNA at two locations and directly switching between them without fully dissociating from the RNA. We develop a biophysical model for ITs in an RNA context that we subsequently validate experimentally. Furthermore, we demonstrate that ITs can differentially accelerate the search process for a broad class of RNA-binding proteins, as opposed to their DNA-binding counterparts, due to RNA’s short persistence length and the heterogeneity of RNA lengths in the cell. Our results illuminate how Cas13 achieves rapid target recognition in a complex RNA environment, and implicate ITs as a potentially widespread solution to the RNA search problem.

Transposon mutagenesis is a powerful method to create deep libraries of genetically diverse cells. However, it has previously not been possible to analyze transposon libraries with respect to complex phenotypes as characterized by intracellular spatial dynamics. Here, we use optical pooled screening to characterize a transposon mutagenesis library via live cell single-particle imaging. The library is analyzed in real time, which allows us to use an optical tweezer to isolate cells with interesting phenotypes. We used the method to identify mutants with perturbations in replication initiation control in Escherichia coli, but it can in principle be used to identify genetic elements associated with any type of complex or dynamic single-cell phenotype.

The ribosome's DNA-encoded production of defined polymer sequences is naturally limited to 22 amino acids. Although the translation machinery has the latent capacity to polymerize backbone-modified substrates, including β-amino acids, this potential is constrained by the intrinsic α-selectivity of native aminoacyl-tRNA synthetases. Here, we address this limitation by "reverse engineering" the E. coli protein ligase EpmA. Naturally activating (R)-β-lysine, EpmA evolved to discard its tRNA-binding domain in favor of protein recognition. By grafting the anticodon-binding domain of the canonical lysyl-tRNA synthetase, LysRS, onto EpmA, we created the chimeric enzyme chEpmA. To our knowledge, this represents the first successful reprogramming of a protein ligase into a functional aminoacyl-tRNA synthetase. We demonstrate that chEpmA serves as a versatile dual-specificity platform: it efficiently charges tRNAs with the non-canonical backbone (R)-β-lysine, and a single substitution unlocks the scaffold for α-substrates, thereby enabling a broad spectrum of post-translational modifications previously inaccessible to genetic code expansion. This repertoire ranges from acylated lysines such as Nε-succinyl-(S)-α-lysine (Ksucc) and bulky modifications such as biocytin to advanced glycation end products (AGEs) including Nε-carboxymethyl-(S)-α-lysine (CML). Our work establishes a structural blueprint for mobilizing non-canonical substrates, paving the way for the biosynthesis of protease-resistant peptidomimetics and next-generation therapeutics.

The corn smut fungus Ustilago maydis is an important microbial model organism representing a genetically amenable and readily cultivable basidiomycete. Research in this fungus addresses a broad range of fundamental questions and its biotechnological exploitation is on the rise. Although genetic engineering in principle is well established, efficient methodology for synthetic biology approaches such as metabolic engineering or pathway transplantation has remained limited. Here, we present a comprehensive toolbox for U. maydis based on modular cloning and the characterization of more than 20 promoters. Careful comparative evaluation of insertion loci and terminator as well as reporter effects was conducted and a novel color-based strategy for straightforward genome integration was implemented. Moreover, the cloning and subsequent one-step integration of four transcriptional units into U. maydis was demonstrated by creating a “rainbow” strain producing four fluorescent proteins. Overall, this next generation toolkit strongly advances genetic engineering and systems biology approaches in U. maydis, fostering its development into a valuable and competitive fungal chassis and prime model, particularly in applied research.

Conjugative plasmids are major drivers of antibiotic resistance dissemination, yet how newly transferred plasmids establish in recipient cells remains poorly understood. Here we investigate YfjB, a previously uncharacterized conserved leading-region protein, which is zygotically induced immediately after plasmid entry and acts specifically during the earliest post-transfer stages. Multi-omics analyses reveal that YfjB reprograms host transcription, triggering extensive metabolic rewiring that compensates for the transient fitness cost of plasmid acquisition. Structural analyses show that YfjB is a ParB-like protein containing a CTP-binding domain and a helix-turn-helix DNA-binding motif, linked to a previously uncharacterized dimerization module that forms a V-shaped clamp-like architecture compatible with DNA loading. Consistently, live-cell imaging reveals nucleoid-associated foci in transconjugants, and ChIP-seq identifies multiple chromosomal binding sites. We therefore rename the protein HerB (Host Expression Reprogrammer, ParB-like). More broadly, our findings reveal how mobile genetic elements facilitate their dissemination by transiently subverting host physiology.

Bacterial microcompartments (BMCs) are a diverse and widespread class of self-assembling protein-based organelles consisting of a semipermeable protein shell encapsulating an enzymatic core. Isolated BMC shell proteins have been shown to assemble into alternative superstructures such as flat sheets and nanotubes. The self-assembly and modularity of BMC shell proteins make them of great interest as modular platforms for applications involving scaffolding, immobilization, and compartmentalization. While the assembly of BMC shell proteins into higher-order structures has been well-studied, the design of controllable and modular cargo loading is underdeveloped in comparison. Recently, we reported the pH-controlled assembly of CcmK2, the major hexameric shell protein of the β-carboxysome BMC, into monodisperse mesh-like microscale particles. Here, we develop a suite of encapsulation strategies for stochastic or targeted loading of various cargos, as well as the direct conjugation of cargo to CcmK2 particles. Our systematic analysis demonstrates that cargo loading and particle assembly can be modulated by the choice of recruitment strategy and the order of cargo introduction. Our findings also reveal a cooperative cargo loading mechanism during assembly that influences particle sizing and apparent morphology. Our study serves as a blueprint for the rational design of tunable cargo loading into engineered BMC-derived microcompartment systems for diverse biotechnological applications.