Revisiting the central dogma one molecule at a time

The faithful relay and timely expression of genetic information depend on specialized molecular machines, many of which function as nucleic acid translocases. The emergence over the last decade of single-molecule fluorescence detection and manipulation techniques with nm and ? resolution and their application to the study of nucleic acid translocases are painting an increasingly sharp picture of the inner workings of these machines, the dynamics and coordination of their moving parts, their thermodynamic efficiency, and the nature of their transient intermediates. Here we present an overview of the main results arrived at by the application of single-molecule methods to the study of the main machines of the central dogma.     

Unraveling helicase mechanisms one molecule at a time

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo.     

Mechanical biochemistry of proteins one molecule at a time

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work.     

Finishing mitosis, one step at a time

The final stages of mitosis begin in anaphase, when the mitotic spindle segregates the duplicated chromosomes. Mitotic exit is then completed by disassembly of the spindle and packaging of chromosomes into daughter nuclei. The successful completion of mitosis requires that these events occur in a strict order. Two main mechanisms govern progression through late mitosis: dephosphorylation of cyclin-dependent kinase (Cdk) substrates and destruction of the substrates of the anaphase-promoting complex (APC). Here, we discuss the hypothesis that the order of late mitotic events depends, at least in part, on the order in which different Cdk and APC substrates are dephosphorylated or destroyed, respectively.     

Surveying the epigenomic landscape, one base at a time

Epigenomics, the determination of epigenetic landscapes on a genome-wide scale, has progressed at an astounding rate over the past decade. Recent technological developments have enabled base-pair resolution of various epigenomic features, leading to new insights into epigenetic regulation.     

Thermodynamics of RNA melting,one base pair at a time

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe “microscopic” enthalpy–entropy compensation—often seen “macroscopically” across a series of related molecular species—across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy–entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.     

Microtubule polymerization: one step at a time

The dynamic assembly of microtubules is a key factor in many of their functions in the cell and recent experiments give new insight into this process at the molecular level.     

Cancer genomics: one cell at a time

The study of single cancer cells has transformed from qualitative microscopic images to quantitative genomic datasets. This paradigm shift has been fueled by the development of single-cell sequencing technologies, which provide a powerful new approach to study complex biological processes in human cancers.