An integrated perspective on RNA aptamer ligand-recognition models : clearing muddy waters

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.PostprintPeer reviewe

Association of acculturation with cardiac structure and function among Hispanics/Latinos: a cross-sectional analysis of the echocardiographic study of Latinos.

OBJECTIVE:Hispanics/Latinos, the largest immigrant population in the USA, undergo the process of acculturation and have a large burden of heart failure risk. Few studies have examined the association of acculturation on cardiac structure and function. DESIGN:Cross-sectional. SETTING:The Echocardiographic Study of Latinos. PARTICIPANTS:1818 Hispanic adult participants with baseline echocardiographic assessment and acculturation measured by the Short Acculturation Scale, nativity, age at immigration, length of US residence, generational status and language. PRIMARY AND SECONDARY OUTCOME MEASURES:Echocardiographic assessment of left atrial volume index (LAVI), left ventricular mass index (LVMI), early diastolic transmitral inflow and mitral annular velocities. RESULTS:The study population was predominantly Spanish-speaking and foreign-born with mean residence in the US of 22.7 years, mean age of 56.4 years; 50% had hypertension, 28% had diabetes and 44% had a body mass index &gt;30 kg/m2. Multivariable analyses demonstrated higher LAVI with increasing years of US residence. Foreign-born and first-generation participants had higher E/e' but lower LAVI and e' velocities compared with the second generation. Higher acculturation and income &gt;20K were associated with higher LVMI, LAVI and E/e' but lower e' velocities. Preferential Spanish-speakers with an income <20K had a higher E/e'. CONCLUSIONS:Acculturation was associated with abnormal cardiac structure and function, with some effect modification by socioeconomic status

Asymmetric base-pair opening drives helicase unwinding dynamics

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix

Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

Funding: Wellcome Trust Investigator Awards (098391/Z/12/Z and 217196/Z/19/Z) and Cancer Research UK Programme grant (C434/A21747) to R.T.H., Wellcome Trust Studentship (109113/Z/15/Z) to P.M., Wellcome Trust Collaborative Award (215539) and multiuser equipment grant (104833) to S.J.M. Additionally J.C.P. thanks the Scottish Universities Physics Alliance (SUPA) and the University of St. Andrews for financial support.The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.Publisher PDFPeer reviewe

Single molecule spectroscopy of polyfluorene chains reveals β-phase content and phase reversibility in organic solvents

Conjugated polymers are an important class of organic semiconductors that can be deposited from solution to make optoelectronic devices. Among them, poly(9,9′-dioctylfluorene) (PFO) has distinctive optical properties arising from its ability to adopt an ordered planar conformation (β phase) from a disordered glassy phase (α phase). The β phase has attractive optical properties, but the precise mechanism of its formation in solution remains unknown. Here, we have combined specifically tailored polymer synthesis and surface-passivation strategies to provide the first spectroscopic characterization of single PFO chains in solution at room temperature. By anchoring PFO molecules at one end on an anti-adherent surface, we show that isolated chains can adopt the β-phase conformation in a solvent-dependent manner. Furthermore, we find that individual PFO chains can reversibly switch multiple times between phases in response to solvent-exchange events. The methodology presented here for polymer synthesis and immobilization is widely applicable to investigate other luminescent polymers.PostprintPostprintPeer reviewe

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a “ratchet” for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by “reeling” ssDNA

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The photophysical behavior of conjugated polymers used in modern optoelectronic devices is strongly influenced by their structural dynamics and conformational heterogeneity, both of which are dependent on solvent properties. Single molecule studies of these polymer systems embedded in a host matrix have proven to be very powerful to investigate the fundamental fluorescent properties. However, such studies lack the possibility of examining the relationship between conformational dynamics and photophysical response in solution, which is the phase from which films for devices are deposited. By developing a synthetic strategy to incorporate a biotin moiety as a surface attachment point at one end of a polyalkylthiophene, we immobilize it, enabling us to make the first single molecule fluorescence measurements of conjugated polymers for long periods of time in solution. We identify fluctuation patterns in the fluorescence signal that can be rationalized in terms of photobleaching and stochastic transitions to reversible dark states. Moreover, by using the advantages of solution-based imaging, we demonstrate that the addition of oxygen scavengers improves optical stability by significantly decreasing the photobleaching rates.<br/

PCNA and XPF cooperate to distort DNA substrates

XPF is a structure-specific endonuclease that preferentially cleaves 3′ DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3′-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90° kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed

Family Environment and the Metabolic Syndrome: Results from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Sociocultural Ancillary Study (SCAS)

Metabolic syndrome (MetS) is a risk factor for cardiovascular disease. Very limited work has evaluated associations of sociocultural processes with prevalence of the MetS

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents

Funding: EPSRC (EP/P030017/1).The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.Publisher PDFPeer reviewe