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Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron Tomography
314a
Tuesday, February 14, 2017
equilibrium from a non-assembling to an assembling Gag state, providing an advantage for nucleation of assembly only on gRNA. 1542-Plat Kinetics of dCas9 Target Search in Escherichia Coli Daniel Jones, Cecilia Unoson, Prune Leroy, Vladimir Curic, Johan Elf. Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded oligonucleotide? To address this question we study the CRISPR-associated protein Cas9 which can be programmed by guide RNAs to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive dCas9 in living Escherichia coli by combining single molecule fluorescence microscopy and bulk restriction protection assays. We find that it takes a single dCas9 ~100 h to find and bind a specific target, in stark contrast to transcription factors such as LacI, which takes 5 minutes to locate its target. Thus, the price dCas9 pays for flexibility in targeting is time. We further identify a likely role for short-range (20-40) 1D sliding along DNA in dCas9 target search. The physical limitations for Cas9 likely generalize to all systems that are programmed by single stranded oligonucleotides to locate sequences in dsDNA, such as the homologous repair machinery. 1543-Plat Argonaute Target Search is Facilitated by Long Distance Diffusion Tao Ju Cui1, Stanley D Chandradoss1, Jorrit Hegge2, John van der Oost2, Chirlmin Joo1. 1 Delft University of Technology, Delft, Netherlands, 2Wageningen University, Wageningen, Netherlands. Argonaute proteins have a variety of functions ranging from post transcriptional gene silencing in eukaryotes to host defence in prokaryotic systems. Despite the well-established functional understanding of Argonautes, the biophysical understanding of its targeting mechanism remains limited. How is it able to find a specific nucleic acid sequence among numerous decoys? Here we make use of the high spatiotemporal resolution of single-molecule FRET to elucidate the target search mechanism of a mesophilic bacterial Argonaute. We find that it makes use of lateral diffusion for rapid sampling of adjacent sequences while using three-dimensional diffusion in order to cover larger distances efficiently. Similar results have been obtained for thermophilic bacterial Argonaute. The long-distance facilitated diffusion mechanism might be a conserved feature across different Argonaute families and may also be a general feature for other nucleic-acid guide directed nucleoproteins such as CRISPR systems. 1544-Plat How Conformational Dynamics Influences the Protein Search for Targets on DNA Maria P. Kochugaeva, Alexey A. Shvets, Anatoly B. Kolomeisky. Chemistry, Rice University, Houston, TX, USA. Protein search and association to specific target sequences on DNA is essential for all fundamental biological processes. The detailed qualitative and quantitative mechanism of fast target finding is still unknown in spite of numerous experimental and theoretical efforts. Particularly, the role of protein conformational fluctuations in the search dynamics remains uncovered. We extend developed earlier theoretical method to analyze how the conformational dynamic affects the process of finding the specific targets on DNA. Our approach is based on discrete-state stochastic model that takes into account main physical-chemical processes. This allows us to evaluate explicitly the protein search for the targets on DNA at different conditions. Our calculations show that conformational fluctuations might strongly affect the protein search dynamics. We discuss the contribution of the shift in the conformational equilibrium in the target search kinetics. We utilized extensive Monte Carlo computer simulations to validate our theoretical predictions. 1545-Plat Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells Caitlin Davis1, Irisbel Guzman2, David Gnutt3, Martin Gruebele1. 1 Physics, University of Illinois, Urbana, IL, USA, 2Biochemistry, University of Illinois, Urbana, IL, USA, 3University of Illinois, Urbana, IL, USA. While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and
binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners. 1546-Plat Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron Tomography Chunyan Wang1, Jiagang Tu1, Bo Hu1, Ian Molineux2, Jun Liu1. 1 The University of Texas Medical School at Houston, Houston, TX, USA, 2 The University of Texas at Austin, Austin, TX, USA. For successful infection, bacteriophages must overcome multiple barriers in the bacterial cell envelope to translocate viral DNA and proteins into the host cell. However, the molecular mechanisms underlying the phage infection initiation remain poorly understood. Here, we use cryo-electron tomography of Salmonella cells infected by phage P22 to capture intermediates during infection. P22 particle initially attaches to the cell surface obliquely through interactions between phage tail spikes and bacterial lipopolysaccharides. Subsequently the phage needle penetrates the host membrane. Three ejection proteins (gp7, gp16, gp20), which are originally stored in capsid, are ejected and assembled to create a 40 nm long trans-envelope channel between the phage tail and the cytoplasmic membrane. The novel channel serves as the conduit for genome translocation. Using mutant particles lacking one or more of the ejection proteins, our in situ structural analysis demonstrates that gp7 forms an extra-cellular channel, extending from the phage tail to the outer membrane; gp20 forms a mushroom-like structure spanning the periplasm, while gp16 is involved in channel formation in the cytoplasmic membrane. Together with atomic resolution information from mature particles and their proteins, our studies reveal a series of key intermediate infection structures at unprecedented detail. They also unveil a massive remodeling of both the infecting phage and the bacterial cell envelope. The structures we obtain provide the first direct evidence illuminating the functions of the three ejection proteins and their distinct role in phage genome delivery. 1547-Plat Using Site Specific Fluorescent Probes to Examine Replication Fork Destabilization by Regulatory Proteins of the Bacteriophage T4 DNA Replication Complex Davis Jose1, Miya Mary Michael2, Wonbae Lee3, Thomas H. Steinberg2, Andrew H. Marcus3, Peter H. von Hippel1. 1 Institute of Molecular Biology and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA, 2Institute of Molecular Biology, University of Oregon, Eugene, OR, USA, 3Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA. The reconstituted T4 DNA replication complex serves as the simplest model system to examine the ‘core’ replication mechanisms of higher organisms. In this study we used site-specifically introduced fluorescent base analogues to track local conformational changes of the nucleic acid bases, as well as Cy3/ Cy5 dye-pairs inserted into the sugar-phosphate backbones to monitor backbone motions at defined positions within the DNA during interactions of the fork with regulatory proteins. By combining low energy circular dichroism (CD) and fluorescent measurements of the site-specifically introduced fluorescent base analogues with single molecule (sm) FRET experiments with cyanine dyes, we have monitored the binding stoichiometries and interactions of the regulatory proteins with model replication fork constructs. The assembly pathways and binding stoichiometries of gp59 (the helicase loader protein), gp41 (the hexameric helicase) and gp61 (the primase) were studied using functional assays as well as single molecule imaging experiments. The results showed that the stoichiometry of gp59/gp41/gp61 subunits within the functional complex is 1:6:1. CD and fluorescence experiments with base analogue probes show that gp59 preferentially binds to and perturbs the bases at the junction of a forked DNA construct, and that the addition of the gp41 helicase hexamer extends this conformational perturbation deep into the DNA duplex. In contrast, the addition of gp61 perturbs the bases at the fork junction, but not base-pairs
Tuesday, February 14, 2017
equilibrium from a non-assembling to an assembling Gag state, providing an advantage for nucleation of assembly only on gRNA. 1542-Plat Kinetics of dCas9 Target Search in Escherichia Coli Daniel Jones, Cecilia Unoson, Prune Leroy, Vladimir Curic, Johan Elf. Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded oligonucleotide? To address this question we study the CRISPR-associated protein Cas9 which can be programmed by guide RNAs to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive dCas9 in living Escherichia coli by combining single molecule fluorescence microscopy and bulk restriction protection assays. We find that it takes a single dCas9 ~100 h to find and bind a specific target, in stark contrast to transcription factors such as LacI, which takes 5 minutes to locate its target. Thus, the price dCas9 pays for flexibility in targeting is time. We further identify a likely role for short-range (20-40) 1D sliding along DNA in dCas9 target search. The physical limitations for Cas9 likely generalize to all systems that are programmed by single stranded oligonucleotides to locate sequences in dsDNA, such as the homologous repair machinery. 1543-Plat Argonaute Target Search is Facilitated by Long Distance Diffusion Tao Ju Cui1, Stanley D Chandradoss1, Jorrit Hegge2, John van der Oost2, Chirlmin Joo1. 1 Delft University of Technology, Delft, Netherlands, 2Wageningen University, Wageningen, Netherlands. Argonaute proteins have a variety of functions ranging from post transcriptional gene silencing in eukaryotes to host defence in prokaryotic systems. Despite the well-established functional understanding of Argonautes, the biophysical understanding of its targeting mechanism remains limited. How is it able to find a specific nucleic acid sequence among numerous decoys? Here we make use of the high spatiotemporal resolution of single-molecule FRET to elucidate the target search mechanism of a mesophilic bacterial Argonaute. We find that it makes use of lateral diffusion for rapid sampling of adjacent sequences while using three-dimensional diffusion in order to cover larger distances efficiently. Similar results have been obtained for thermophilic bacterial Argonaute. The long-distance facilitated diffusion mechanism might be a conserved feature across different Argonaute families and may also be a general feature for other nucleic-acid guide directed nucleoproteins such as CRISPR systems. 1544-Plat How Conformational Dynamics Influences the Protein Search for Targets on DNA Maria P. Kochugaeva, Alexey A. Shvets, Anatoly B. Kolomeisky. Chemistry, Rice University, Houston, TX, USA. Protein search and association to specific target sequences on DNA is essential for all fundamental biological processes. The detailed qualitative and quantitative mechanism of fast target finding is still unknown in spite of numerous experimental and theoretical efforts. Particularly, the role of protein conformational fluctuations in the search dynamics remains uncovered. We extend developed earlier theoretical method to analyze how the conformational dynamic affects the process of finding the specific targets on DNA. Our approach is based on discrete-state stochastic model that takes into account main physical-chemical processes. This allows us to evaluate explicitly the protein search for the targets on DNA at different conditions. Our calculations show that conformational fluctuations might strongly affect the protein search dynamics. We discuss the contribution of the shift in the conformational equilibrium in the target search kinetics. We utilized extensive Monte Carlo computer simulations to validate our theoretical predictions. 1545-Plat Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells Caitlin Davis1, Irisbel Guzman2, David Gnutt3, Martin Gruebele1. 1 Physics, University of Illinois, Urbana, IL, USA, 2Biochemistry, University of Illinois, Urbana, IL, USA, 3University of Illinois, Urbana, IL, USA. While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and
binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners. 1546-Plat Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron Tomography Chunyan Wang1, Jiagang Tu1, Bo Hu1, Ian Molineux2, Jun Liu1. 1 The University of Texas Medical School at Houston, Houston, TX, USA, 2 The University of Texas at Austin, Austin, TX, USA. For successful infection, bacteriophages must overcome multiple barriers in the bacterial cell envelope to translocate viral DNA and proteins into the host cell. However, the molecular mechanisms underlying the phage infection initiation remain poorly understood. Here, we use cryo-electron tomography of Salmonella cells infected by phage P22 to capture intermediates during infection. P22 particle initially attaches to the cell surface obliquely through interactions between phage tail spikes and bacterial lipopolysaccharides. Subsequently the phage needle penetrates the host membrane. Three ejection proteins (gp7, gp16, gp20), which are originally stored in capsid, are ejected and assembled to create a 40 nm long trans-envelope channel between the phage tail and the cytoplasmic membrane. The novel channel serves as the conduit for genome translocation. Using mutant particles lacking one or more of the ejection proteins, our in situ structural analysis demonstrates that gp7 forms an extra-cellular channel, extending from the phage tail to the outer membrane; gp20 forms a mushroom-like structure spanning the periplasm, while gp16 is involved in channel formation in the cytoplasmic membrane. Together with atomic resolution information from mature particles and their proteins, our studies reveal a series of key intermediate infection structures at unprecedented detail. They also unveil a massive remodeling of both the infecting phage and the bacterial cell envelope. The structures we obtain provide the first direct evidence illuminating the functions of the three ejection proteins and their distinct role in phage genome delivery. 1547-Plat Using Site Specific Fluorescent Probes to Examine Replication Fork Destabilization by Regulatory Proteins of the Bacteriophage T4 DNA Replication Complex Davis Jose1, Miya Mary Michael2, Wonbae Lee3, Thomas H. Steinberg2, Andrew H. Marcus3, Peter H. von Hippel1. 1 Institute of Molecular Biology and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA, 2Institute of Molecular Biology, University of Oregon, Eugene, OR, USA, 3Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA. The reconstituted T4 DNA replication complex serves as the simplest model system to examine the ‘core’ replication mechanisms of higher organisms. In this study we used site-specifically introduced fluorescent base analogues to track local conformational changes of the nucleic acid bases, as well as Cy3/ Cy5 dye-pairs inserted into the sugar-phosphate backbones to monitor backbone motions at defined positions within the DNA during interactions of the fork with regulatory proteins. By combining low energy circular dichroism (CD) and fluorescent measurements of the site-specifically introduced fluorescent base analogues with single molecule (sm) FRET experiments with cyanine dyes, we have monitored the binding stoichiometries and interactions of the regulatory proteins with model replication fork constructs. The assembly pathways and binding stoichiometries of gp59 (the helicase loader protein), gp41 (the hexameric helicase) and gp61 (the primase) were studied using functional assays as well as single molecule imaging experiments. The results showed that the stoichiometry of gp59/gp41/gp61 subunits within the functional complex is 1:6:1. CD and fluorescence experiments with base analogue probes show that gp59 preferentially binds to and perturbs the bases at the junction of a forked DNA construct, and that the addition of the gp41 helicase hexamer extends this conformational perturbation deep into the DNA duplex. In contrast, the addition of gp61 perturbs the bases at the fork junction, but not base-pairs