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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., Eukaryotic DNA replication is regulated to ensure copying of the genome (only) once per cell cycle. Here the authors, using optical trapping and confocal microscopy, demonstrate the dynamics of the origin recognition complex and subsequent intermediates that lead up to the loading of an MCM helicase onto DNA.

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Forster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.

RNA folding studies aim to clarify the relationship among sequence, tridimensional structure, and biological function. In the last decade, the application of single-molecule fluorescence resonance energy transfer (sm-FRET) techniques to investigate RNA structure and folding has revealed the details of conformational changes and timescale of the process leading to the formation of biologically active RNA structures with subnanometer resolution on millisecond timescales. In this review, we initially summarize the first wave of single-molecule FRET-based RNA techniques that focused on analyzing the influence of mono- and divalent metal ions on RNA function, and how these studies have provided very valuable information about folding pathways and the presence of intermediate and low-populated states. Next, we describe a second generation of single-molecule techniques that combine sm-FRET with the use of chemical denaturants as an emerging powerful approach to reveal information about the dynamics and energetics of RNA folding that remains hidden using conventional sm-FRET approaches. The main advantages of using the competing interplay between folding agents such as metal ions and denaturants to observe and manipulate the dynamics of RNA folding and RNA-ligand interactions is discussed in the context of the adenine riboswitch aptamer.

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 gene-regulatory RNA motifs located in the 5' untranslated regions of certain bacterial mRNA's. Riboswitches regulate gene expression by binding a metabolite related to the downstream gene, causing a conformational change that alters the accessibility of the gene for either transcription or translation. An important class of riboswitches that bind fluoride (F-) has been identified recently in bacteria and archaea, shedding light on how these organisms regulate internal fluoride concentrations to mitigate toxicity. The crystal structure of the fluoride riboswitch from Thermophilus petrophila shows a binding pocket in which the F- ion is coordinated by three Mg2+ ions. However, how ligand recognition and RNA folding are coupled to selectively encapsulate F- is not fully understood. Here, single-molecule TIRF microscopy and FRET are used to gain insights into the functional dynamics of fluoride riboswitches. Individual fluorescently-labelled fluoride riboswitches are immobilized on a quartz microscope slide, and the change in FRET efficiency between the fluorophors is used to study the ligand-binding mechanism and other cation- or denaturant-dependent structural transitions.