Dr Tara Sabir

Branched structures of nucleic acids are widely observed in nature as intermediates during DNA repair, recombination and replication processes, as well as being a component of vital structures for protein synthesis. Using highresolution single-molecule Fo╮ster resonance energy transfer (SM-FRET), we showed recently that a DNA three way junction was not fully paired at the branchpoint, in spite of being fully complementary.(1) To investigate whether this expanded branchpoint was a general effect, or due to the specific sequence used, we have also studied a three-way junction with a GC-rich branchpoint. We report the results of SM-FRET and ensemble NMR experiments on this new junction, together with initial investigations of the branchpoint reactivity. 1. Sabir, Tara et al. "Branchpoint Expansion in a Fully Complementary ThreeWay DNA Junction." J. Am. Chem. Soc. (2012) 134: 6280 6285

Branched DNA structures play critical roles in DNA replication, repair, and recombination in addition to being key building blocks for DNA nanotechnology. Here we combine single-molecule multiparameter fluorescence detection and molecular dynamics simulations to give a general approach to global structure determination of branched DNA in solution. We reveal an open, planar structure of a forked DNA molecule with three duplex arms and demonstrate an ion-induced conformational change. This structure will serve as a benchmark for DNA-protein interaction studies.

As educators in biomedical science, translating complex theory formulates how a module is designed, assessed and presented. This leaves little time to ensure that included within that translation are the realities of working for commercial labs or even the National Health Service post qualification. Authentic learning experiences can be hard to achieve within the physical constraints of university education, especially within the biosciences. One of the biggest challenges faced when designing university courses is to replicate the time pressures associated with a professional laboratory setting. It is critical for students entering into careers within this competitive and high-pressure field to understand the expectations of their employers. Authentic experiences are the only way to ensure that the realities of scientific practice and professionalism hit home to those students looking to gain future employment. This chapter describes the authentic learning experiences employed mid-degree for biomedical science students, drawing upon the importance of personal responsibility, bioethics and the importance of data trailing, protocol and experimental design as well as essential calculations, risk assessments and health and safety. This aims to prepare graduates with a foundation of core skills to gain employment in their first professional post within industry or academia.

Abstract

The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster ( ∼ 20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric coupling in which SecA ‘steers’ the energy landscape of the protein-channel.

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10

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DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first (19)F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.