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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