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.

Stochastic Analysis Demonstrates the Dual Role of Hfq in Chaperoning 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 . The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation.

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.

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.

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.