No abstract

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here, we show that conformational control through intramolecular crosslinking of a helicase monomer with undetectable unwinding activity converts it into a super-helicase that can unwind thousands of base pairs processively even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into how nature achieves the regulation of nucleic acid unwinding activity and introduces a monomeric super-helicase without nuclease activities which may be useful for biotechnological applications.

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.DOI: http://dx.doi.org/10.7554/eLife.08193.001

Multidrug resistant organisms (MDROs) are a serious threat to human health 1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying MDROs increase mortality 3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution 5 , require several days before informing key clinical decisions. Rapid AST would transform the care of infected patients while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here, we describe a rapid assay for combined Genotypic and Phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology, and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 hours faster than standard workflows, with <4 hour assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.