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SUMMARY:Light\, camera\, action: watching single DNA/RNA polymerases at wo
 rk in living cells - Achillefs Kapanidis\, Dept Physics\, University of Ox
 ford
DTSTART:20131129T140000Z
DTEND:20131129T150000Z
UID:TALK48134@talks.cam.ac.uk
CONTACT:Dr Eileen Nugent
DESCRIPTION:Most single-molecule experiments are performed in vitro\, usin
 g tightly controlled conditions and well-defined concentrations of a limit
 ed number of interacting components. However\, to understand biological me
 chanisms as they occur in vivo while maintaining the extra information pro
 vided by single-molecule detection\, there is a need for performing such e
 xperiments in cellular contexts\, and in particular in living cells.\n\nTo
 wards this goal\, we have been single-molecule fluorescence to perform loc
 alization-based super-resolution imaging (specifically photoactivated loca
 lization microscopy\, PALM) and to study the subcellular localization\, di
 ffusion properties\, abundance and clustering of DNA-binding proteins in b
 acterial cells. We have also developed physical methods for delivering flu
 orescent biomolecules in bacteria and observing single-molecule fluorescen
 ce and single-molecule FRET in the bacterial cytoplasm. \n\nI will discuss
  PALM studies of the subcellular localization and copy number of RNA polym
 erase in bacterial cells grown under different physiological conditions\, 
 both in fixed and living bacterial cells\; using this method\, we have ide
 ntified the spatial distribution of an immobile fraction that represents m
 ainly RNAP polymerases engaged in promoter and transcribed regions. \n\nI 
 will also how describe how PALM-based single-molecule diffusion tracks of 
 polymerases in living bacteria (middle and bottom panels) allowed us to vi
 sualize DNA-damage and DNA-repair processes in real-time and at the single
 -molecule level for the first time. \n\nOur approaches should be useful fo
 r studying many processes in gene replication\, expression\, and maintenan
 ce in various types of cells.\n\n\n
LOCATION:Small Lecture Theatre\, Cavendish Laboratory
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