Group meeting

Postdoc interview

  • Albert Tsai
  • Observing translation in real-team: tracking prokaryotic translation with single molecule fluorescence.
  • Jody Puglisi (Stanford)

Introduction

  • ribosome (20×25 nm)
  • translation: many stochastic and cyclical substeps. Difficult to synchronize
  • large number of possible states.
  • time resolution 10ms.

Project 1: initiation pathway:

  • from separate ribosome subunits to elongating ribosome
  • 30s pre-initiation complex (PIC). Find shine-delgarno sequence in mRNA, initiator tRNA binds, GTPase initiation factor 2 stabilizes complex.
  • look at arrival and departure of dye labeled components
  • Challenge: need high ligand concentration to have fast dynamics (reaction before bleaching)
  • solution: add zero-mode waveguides: 100 nm laser quenching modificaitons create wells on surface. (originally developed for high-throughput sequencing)
  • few hundred analyzable molecules per field of view.
  • sometimes IF2 first, sometimes tRNA first, sometimes both in same frame. Substantially higher than expected simultaneous arrival (given individual arrival rates 1-2s (each) and frame-rate 10-100 per second) –> preformed complex.
  • preformed complex in vivo.
  • Forming the 70S complex
  • IF2 ‘gates’ tRNA arrival, IF2 leaves after 50S joins.
  • IF2 departure rate limiting single step, tRNA concentration independent 2s.
  • Any new insights into mRNA sequence dependent regulation? –> Other regulatory factors also involved in sequence dependent effects. Examined different Shine-Delgarno sequences but not in detail.
  • see article Nature 2012.

Project 2: elongation:

  • 1 GTP used per step (tRNA arrival and ribosome translocation)
  • balance of speed and accuracy.
  • target of many antibiotics
  • e.g. paromomycin structure suggests it stabilizes tRNA independent of mRNA – tRNA codon match, leading to increase error rate.
  • use FRET between ribosome subunits to track ribosome confirmation movements
  • count FRET cycles as a measure of elongation speed.
  • with drugs, most ribosomes “stop” early — fewer cycles (see article Cell Reports 2013).
  • is it making fewer cycles or just going slower and eventually photobleaching
  • individual step kinetics are slower (some drugs just effect 1 of the steps, some just effect at first codon).
  • does apromycin inhibit EFG binding (GTP delivery). binding frequency of EFG doesn’t really change, number of binding events before translocation does increase — barrier to translocation step.
  • TIRF SNR vs zero-mode wave guide: not strong difference, maybe TIRF better? XZ thinks zero-mode wave guide with shorter penetration depth should be better.
  • paromomycin and gentamicin slowing down peptide bond formation? – no. block conformational change of ribosome itself (subunit rotation step). Expect kinetic shutdown more important than AA misincorporation.

Project 3:

  • looking at real mRNA sequences
  • bulk charged tRNA (AA loaded) + labeled components. Not using cell extract.
  • SecM stall sequence (waits until ribosome is docked with secretion channel, secrete protein through channel).
  • SecM interacts with 50S exit tunnel (not a passive pipe).
  • observe stalling not at a consistent fixed AA position. Progressively increased lifetime near the stall site leads to eventual stopping.
  • mutate first critical site, no stalling, second critical site, rotated state stalls, no rotated state doesn’t stall.
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