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  • 1
    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
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    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
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
    Publikationsdatum: 2014-05-03
    Beschreibung: Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108260/" 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/PMC4108260/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Larson, Matthew H -- Mooney, Rachel A -- Peters, Jason M -- Windgassen, Tricia -- Nayak, Dhananjaya -- Gross, Carol A -- Block, Steven M -- Greenleaf, William J -- Landick, Robert -- Weissman, Jonathan S -- F32 GM100611/GM/NIGMS NIH HHS/ -- F32 GM108222/GM/NIGMS NIH HHS/ -- P50 GM102706/GM/NIGMS NIH HHS/ -- R01 GM038660/GM/NIGMS NIH HHS/ -- R01 GM102790/GM/NIGMS NIH HHS/ -- R37 GM057035/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2014 May 30;344(6187):1042-7. doi: 10.1126/science.1251871. Epub 2014 May 1.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, California Institute for Quantitative Biosciences, Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. ; Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA. ; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA. ; Department of Biological Sciences, Stanford University, Stanford, CA 94025, USA. Department of Applied Physics; Stanford University, Stanford, CA 94025, USA. ; Department of Genetics, Stanford University, Stanford, CA 94025, USA. wjg@stanford.edu landick@biochem.wisc.edu weissman@cmp.ucsf.edu. ; Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA. Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. wjg@stanford.edu landick@biochem.wisc.edu weissman@cmp.ucsf.edu. ; Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, California Institute for Quantitative Biosciences, Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. wjg@stanford.edu landick@biochem.wisc.edu weissman@cmp.ucsf.edu.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24789973" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Base Sequence ; Codon, Initiator/*genetics ; Consensus Sequence ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/*genetics/*metabolism ; *Gene Expression Regulation, Bacterial ; Peptide Chain Initiation, Translational/*genetics ; *Regulatory Elements, Transcriptional ; *Transcription, Genetic
    Print ISSN: 0036-8075
    Digitale ISSN: 1095-9203
    Thema: Biologie , Chemie und Pharmazie , Informatik , Medizin , Allgemeine Naturwissenschaft , Physik
    Standort Signatur Erwartet Verfügbarkeit
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