Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA.

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.

Stochastic Analysis Demonstrates the Dual Role of Hfq in Chaperoning E. coli Sugar Shock Response.

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or "sugar shock" in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS.

Small RNA (sRNA) regulators promote efficient responses to stress, but the mechanisms for prioritizing target mRNA regulation remain poorly understood. This study examines mechanisms underlying hierarchical regulation by the sRNA SgrS, found in enteric bacteria and produced under conditions of metabolic stress. SgrS posttranscriptionally coordinates a nine-gene regulon to restore growth and homeostasis. An in vivo reporter system quantified SgrS-dependent regulation of target genes and established that SgrS exhibits a clear target preference. Regulation of some targets is efficient even at low SgrS levels, whereas higher SgrS concentrations are required to regulate other targets. In vivo and in vitro analyses revealed that RNA structure and the number and position of base pairing sites relative to the start of translation impact the efficiency of regulation of SgrS targets. The RNA chaperone Hfq uses distinct modes of binding to different SgrS mRNA targets, which differentially influences positive and negative regulation. The RNA degradosome plays a larger role in regulation of some SgrS targets compared to others. Collectively, our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features and that the combination of these features precisely tunes the efficiency of regulation of multi-target sRNA regulons.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.