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  • 1
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    Direct observation of the binding state of the kinesin head to the microtubule (2009)
    Nature Publishing Group (NPG)
    Details
    Publikationsdatum: 2009-08-21
    Beschreibung: The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859689/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859689/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Guydosh, Nicholas R -- Block, Steven M -- GM51453/GM/NIGMS NIH HHS/ -- R01 GM051453/GM/NIGMS NIH HHS/ -- R01 GM051453-15/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Sep 3;461(7260):125-8. doi: 10.1038/nature08259. Epub 2009 Aug 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Biophysics Program, Stanford University, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19693012" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Adenosine Triphosphate/metabolism/pharmacology ; Animals ; DNA/chemistry/metabolism ; Drosophila melanogaster ; Kinesin/*chemistry/*metabolism ; Microspheres ; Microtubules/*metabolism ; Movement/drug effects ; Optical Tweezers ; Protein Binding/drug effects ; Time Factors
    Print ISSN: 0028-0836
    Digitale ISSN: 1476-4687
    Thema: Biologie , Chemie und Pharmazie , Medizin , Allgemeine Naturwissenschaft , Physik
    Publiziert von Nature Publishing Group (NPG)
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
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    Sequence-dependent pausing of single lambda exonuclease molecules (2003)
    American Association for the Advancement of Science (AAAS)
    Details
    Publikationsdatum: 2003-08-30
    Beschreibung: Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539570/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539570/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Perkins, Thomas T -- Dalal, Ravindra V -- Mitsis, Paul G -- Block, Steven M -- GM 57035/GM/NIGMS NIH HHS/ -- HG 011821-01/HG/NHGRI NIH HHS/ -- R01 GM057035/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2003 Sep 26;301(5641):1914-8. Epub 2003 Aug 28.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA. tperkins@jila.colorado.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/12947034" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Bacteriophage lambda/enzymology ; Base Pairing ; *Base Sequence ; Binding Sites ; Consensus Sequence ; DNA/*chemistry/*metabolism ; Electrophoresis, Polyacrylamide Gel ; Exodeoxyribonucleases/*metabolism ; Hydrogen Bonding ; Kinetics ; Models, Chemical ; Oligodeoxyribonucleotides/chemistry/metabolism ; Polymerase Chain Reaction ; Probability ; Stochastic Processes ; Time Factors ; Viral Proteins
    Print ISSN: 0036-8075
    Digitale ISSN: 1095-9203
    Thema: Biologie , Chemie und Pharmazie , Informatik , Medizin , Allgemeine Naturwissenschaft , Physik
    Publiziert von American Association for the Advancement of Science (AAAS)
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
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    Real-time observation of the initiation of RNA polymerase II transcription (2015)
    Nature Publishing Group (NPG)
    Details
    Publikationsdatum: 2015-09-04
    Beschreibung: Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624315/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624315/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Fazal, Furqan M -- Meng, Cong A -- Murakami, Kenji -- Kornberg, Roger D -- Block, Steven M -- AI21144/AI/NIAID NIH HHS/ -- GM36659/GM/NIGMS NIH HHS/ -- GM57035/GM/NIGMS NIH HHS/ -- R01 AI021144/AI/NIAID NIH HHS/ -- R01 GM036659/GM/NIGMS NIH HHS/ -- R01 GM049985/GM/NIGMS NIH HHS/ -- R37 GM057035/GM/NIGMS NIH HHS/ -- England -- Nature. 2015 Sep 10;525(7568):274-7. doi: 10.1038/nature14882. Epub 2015 Sep 2.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Applied Physics, Stanford University, Stanford, California 94305, USA. ; Department of Chemistry, Stanford University, Stanford, California 94305, USA. ; Department of Structural Biology, Stanford University, Stanford, California 94305, USA. ; Department of Biology, Stanford University, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26331540" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Base Pairing/genetics ; DNA/genetics/metabolism ; Optical Tweezers ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/*chemistry/*metabolism ; RNA, Messenger/biosynthesis/genetics ; Saccharomyces cerevisiae/*enzymology/genetics ; Transcription Factor TFIIH/metabolism ; *Transcription Initiation, Genetic
    Print ISSN: 0028-0836
    Digitale ISSN: 1476-4687
    Thema: Biologie , Chemie und Pharmazie , Medizin , Allgemeine Naturwissenschaft , Physik
    Publiziert von Nature Publishing Group (NPG)
    Standort Signatur Erwartet Verfügbarkeit
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  • 4
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    Direct observation of hierarchical folding in single riboswitch aptamers (2008)
    American Association for the Advancement of Science (AAAS)
    Details
    Publikationsdatum: 2008-01-05
    Beschreibung: Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2640945/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2640945/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Greenleaf, William J -- Frieda, Kirsten L -- Foster, Daniel A N -- Woodside, Michael T -- Block, Steven M -- GM057035/GM/NIGMS NIH HHS/ -- GM066275/GM/NIGMS NIH HHS/ -- R01 GM057035/GM/NIGMS NIH HHS/ -- R01 GM057035-12/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2008 Feb 1;319(5863):630-3. doi: 10.1126/science.1151298. Epub 2008 Jan 3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18174398" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Adenine/chemistry/metabolism ; Aptamers, Nucleotide/*chemistry/metabolism ; Base Pairing ; DNA-Directed RNA Polymerases/metabolism ; Ligands ; *Nucleic Acid Conformation ; Optical Tweezers ; RNA, Messenger/*chemistry/metabolism ; Thermodynamics
    Print ISSN: 0036-8075
    Digitale ISSN: 1095-9203
    Thema: Biologie , Chemie und Pharmazie , Informatik , Medizin , Allgemeine Naturwissenschaft , Physik
    Publiziert von American Association for the Advancement of Science (AAAS)
    Standort Signatur Erwartet Verfügbarkeit
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