DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.
We have investigated the effect of Airy illumination on the image quality and depth penetration of digitally scanned light-sheet microscopy in turbid neural tissue. We used Fourier analysis of images acquired using Gaussian and Airy light-sheets to assess their respective image quality versus penetration into the tissue. We observed a three-fold average improvement in image quality at 50 μm depth with the Airy light-sheet. We also used optical clearing to tune the scattering properties of the tissue and found that the improvement when using an Airy light-sheet is greater in the presence of stronger sample-induced aberrations. Finally, we used homogeneous resolution probes in these tissues to quantify absolute depth penetration in cleared samples with each beam type. The Airy light-sheet method extended depth penetration by 30% compared to a Gaussian light-sheet.
Single-molecule fluorescence studies of nucleic acids are revolutionizing our understanding of fundamental cellular processes related to DNA and RNA processing mechanisms. Detailed molecular insights into DNA repair, replication, transcription, and RNA folding and function are continuously being uncovered by using the full repertoire of single-molecule fluorescence techniques. The fundamental reason behind the stunning growth in the application of single-molecule techniques to study nucleic acid structure and dynamics is the unmatched ability of single-molecule fluorescence, and mostly single-molecule FRET, to resolve heterogeneous static and dynamic populations and identify transient and low-populated states without the need for sample synchronization. New advances in DNA and RNA synthesis, post-synthetic dye-labeling methods, immobilization and passivation strategies, improved dye photophysics, and standardized analysis methods have enabled the implementation of single-molecule techniques beyond specialized laboratories. In this chapter, we introduce the practical aspects of applying single-molecule techniques to investigate nucleic acid structure, dynamics, and function.
Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.
Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.
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