Proton-Coupling Bioenergetics Sunday, March 3, 2019
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12a Sunday, March 3, 2019 Symposium: Biological Systems Single Molecule encode hundreds of ribosomal DNA operons (rDNA) that exhibit extensive sequence variation with the rRNA components of the ribosome, which are at the Time both conserved and expressed in a tissue-specific fashion. Using E.coli as a genetically tractable model system, we now show that nutrient limitation- 65-Symp induced stress changes the relative expression of rDNA operons to alter the ri- The Mechanism of Dynein Directionality bosomal RNA (rRNA) composition within the actively translating ribosome Ahmet Yildiz. pool. The most upregulated operon encodes the unique 16S rRNA gene, Physics, Univ. California Berkeley, Berkeley, CA, USA. rrsH, distinguished by conserved sequence variation within the small The ability of cytoskeletal motors to move unidirectionally along filamentous ribosomal subunit. rrsH-bearing ribosomes alter the levels of the RpoS sigma tracks is central to their role in cargo transport, motility and cell division. factor, the master regulator of the general stress response, to affect the expres- While kinesin and myosin motor families have members that move in oppo- sion of functionally coherent gene sets. These impacts are associated with site directions, all dyneins studied to date exclusively move towards the phenotypic changes in antibiotic sensitivity, biofilm formation, and cell microtubule (MT) minus-end. In order to understand the mechanism of motility, and are regulated by ribosome-associated stress response proteins. dynein’s directionality, we sought to engineer a plus-end-directed dynein Specific aspects of these phenotypic differences could be reconstituted in vitro guided by cryo-electron microscopy and molecular dynamics simulations. using highly purified translation components. These findings establish that As shown by single-molecule assays, elongation or shortening of the endogenously encoded, naturally occurring rRNA sequence variation can coiled-coil stalk that connects the motor to the MT controls helical direction- modulate ribosome function, central aspects of gene expression regulation, ality of S. cerevisiae dynein around MTs. By changing the length and angle of and cellular physiology. the stalk, we successfully reversed dynein motility towards the MT plus-end. These modifications act by altering the direction dynein’s linker swings 68-Symp relative to the MT, not by reversing the asymmetric unbinding of the motor Probing the Dynamics and Interactions of Disordered Proteins with Single- from MT. Because the length and angle of dynein’s stalk are fully conserved Molecule Spectroscopy among species, our findings provide an explanation for why all dyneins move Ben Schuler. towards the MT minus-end. University of Zurich, Zurich, Switzerland. The functions of proteins have traditionally been linked to their well-defined 66-Symp three-dimensional, folded structures. It is becoming increasingly clear, how- In Situ Imaging of Transcriptome and Genome in Single Cells ever, that many proteins perform essential functions without being folded. Xiaowei Zhang. Single-molecule spectroscopy provides new opportunities for investigating Chemistry and Chemical Biology, Howard Hughes Medical Institute, the structure and dynamics of such unfolded or ‘intrinsically disordered’ pro- Harvard University, Cambridge, MA, USA. teins (IDPs). The combination of single-molecule Fo¨rster resonance energy In situ transcriptomic analysis of single cells promise to transform our under- transfer (FRET) with nanosecond correlation spectroscopy, microfluidic mix- standing in many areas of biology, such as regulation of gene expression, devel- ing, and other advanced methods can be used to probe intra- and intermolecular opment of cell fate, and organization of distinct cell types in complex tissues. distance distributions, reconfiguration dynamics, and interactions on a wide We developed a single-cell transcriptome imaging method, multiplexed error- range of timescales, and even in heterogeneous environments, including live robust fluorescent in situ hybridization (MERFISH), which uses combinatorial cells. labeling and sequential imaging to massively multiplex single-molecule FISH I will illustrate the use of single-molecule FRET in combination with other bio- measurements and error-robust encoding schemes to minimize measurement physical methods in the context of an unexpected interaction mechanism that error, enabling RNA imaging and profiling at the transcriptomic scale. By we recently discovered: The two intrinsically disordered human proteins his- allowing single-cell transcriptomic analysis in the native context of cells and tone H1 and its nuclear chaperone prothymosin a associate in a one-to-one tissues, MERFISH facilitates the delineation of gene regulatory networks, the complex with picomolar affinity, but they fully retain their structural disorder, mapping of RNA distributions inside cells, and the mapping of distinct cell long-range flexibility, and highly dynamic character. Based on the close inte- types in complex tissues. In this presentation, I will talk about our technology gration of single-molecule experiments, NMR, and molecular simulations, development and recently applications of MERFISH, with a focus on mapping we obtained a detailed picture of this complex that can be explained by the the organization of distinct cell types in the brain. large opposite net charge of the two proteins without requiring defined binding I will also talk about a multiplexed FISH method that we developed for imaging sites or interactions between specific individual residues. This type of interac- the 3D conformation of chromosomes in single cells. The spatial organization tion has interesting ramifications for kinetic mechanisms of binding and of genome plays an important role in many essential genome functions from cellular regulation. gene regulation to genome replication. However, many gaps remain in our un- derstanding of the 3D organization of chromatin in the nucleus, partly because of the lack of proper imaging tools. Our multiplex FISH method allows Symposium: Proton-coupling Bioenergetics numerous genomic loci to be imaged and localized. Using this approach, we traced the 3D conformation of chromatin in cell nucleus with high resolution, 69-Symp C and revealed novel spatial organization of chromatin domains and compart- The Proton/Electron Coupling Mechanism of Cytochrome Oxidase 1 2 ments in individual chromosomes. Peter R. Rich , Vivek Sharma . 1Structural and Molecular Biology, University College London, London, 67-Symp United Kingdom, 2Physics, University of Helsinki, Helsinki, Finland. Endogenously Encoded Ribosomal RNA Sequence Variation within the Mitochondrial cytochrome c oxidases (CcOs) are members of a large haem- Assemble Ribosome can Regulate Stress Response Gene Expression and copper oxidase superfamily. They fall within the A1 branch which includes Phenotype closely structurally-related bacterial CcOs and quinol oxidases. Three possible Scott C. Blanchard1, Chad M. Kurylo1, Matt M. Parks1, Manuel F. Juette1, networks of amino acids and waters have been identified in subunit I for intra- Boris Zinshteyn1, Roger B. Altman1, Theresa C. Vincent1, protein transfer of substrate and translocated protons. The D and K channels are Michael R. Wasserman2, Jose L. Alejo Amaya3, Daniel S. Terry1. very similar in all structures. The H channel was firstly described in bovine 1Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA, CcO, though several of its features are evident bacterial A1-type CcOs. In bac- 2Rockefeller Univ, New York, NY, USA, 3Weill Cornell Med Coll, New terial systems, there is substantial evidence that the D channel provides the York, NY, USA. route for pumped protons to a temporary proton trap site, though the exit route Prevailing dogma holds that ribosomes are uniform in composition and func- into the P phase remains uncertain. In contrast, in mammalian mitochondrial tion. Single-molecule investigations of translation reveal, however, that the CcO structural and functional data have suggested that the H channel may ribosome and translation factors visit metastable, transient intermediates that instead provide the route for pumped protons to a similar trap site location, can be highly sensitive to even modest perturbations, including small- and that its ‘top’ half provides the exit route from the trap into the P phase. molecule drugs and single-nucleotide substitutions in ribosomal RNA Direct measurements of coupling efficiencies of yeast mitochondrial CcO (rRNA). These insights suggest the possibility that subtle changes in the ribo- with mutations in these potential proton pathways showed that pumped protons some’s composition may have functional impacts. Recent evidence further im- are transferred through its D channel. Furthermore, MD simulations indicate plicates changes in the ribosome’s core protein composition with gene-specific that the ‘lower’ region of its H channel could not provide a transfer route changes in translational efficiency. In this light, we set out to examine the po- into the trap site. This is consistent with prior MD simulations of this region tential physiological impacts of endogenously encoded sequence variations in of the bovine CcO H channel which supported a dielectric well, but not a proton the rRNA components of the assembled ribosome. Mammalian genomes transfer, role. However, the ‘top’ part of the H channel may indeed form a facile BPJ 9279_9291 Sunday, March 3, 2019 13a route for