AGBT 2010 - Joseph Puglisi - Stanford University School of Meicine
Questions have made the same, despite recent advances - we still want to understand how the molecular machines work. We always have snapshots that capture the element of motion, but we want animation, not snapshots
- Converting nucleotides to amino acids.
- ribosome 1-20 aa/s
- 1/10^4 errors
- very complex process (tons of proteins factors, etc, required for the process)
-requires micro-molar concentrations of each component
- we now know the structure of the ribosome
- nobel prize given for it.
- 2 subunits. (50S & 30S)
- 3 sites, E, P & A
- image 3 trna's to a ribosome - in the 3 sites...
- all our shots are static - no animated
- The Ribosome selects tRNA for Catalysis - must be correct, and incorrect must be rapidly rejected
- EFTu involved in rejection
[Walking us through how ribosomes work - there are better sources for this on the web, so I'm not going to copy it.]
= timing of factor
- initiation pathway
- origins of translational fidelity
Look at it as a high dynamic process
- flux of tRNAs
- movements of the ribosome (internal and external)
- much slower than photosynthesis, so easier to observe.
Can we track this process in real time?
- Try: Label the ligand involved in translation.
- Problem: solution averaging destroys signal (many copies of ribosome get out of sync FAST.) would require single molecule monitoring
- Solution: immobilization of single molecule - also allows us to watch for a long time
Single molecule real time translation
- Functional fluorescent labeling of tRNAs ribosomes and factors
- surface immobilization retains function.
- observation of translation at micromolar conc. fluorescent components
- instrumentation required to resolve multiple colors
- yes, it does work.
- you can tether with biotin-streptavidin, instead of fixing to surface
- immobilization does not modify kinetics
Tried this before talking to Pac Bio - It was a disaster. Worst experiments they'd ever tried.
- use PAcBio ZMW to do this experiment.
- has multiple colour resolution required
- 10ms time resolution
Can you put a 20nm ribosome into a 120nm hole? Use biotin tethering - Yes
Can consecutive tRNA binding be observed in real time? Yes
Flourescence doesn't leave after... they overlap because the labeled tRNA must transit through the ribosome.
- at low nanomolar sigals, you can see the signals move through individual
- works at higher conc.
- if you leave EF-G out, you get binding, but no transit - then photobleaching.
- demonstrate Lys-tRNA
- 3 three labeled dyes (M, F, K)... you can see it work.
- timing isn't always the same (pulse length)
-missing stop coding - so you see really long stall with labeled dye... and then sampling, as other tRNAs try to fit.
- you can also sequence as you code. [neat]
Decreased tRNA transit time at higher EF-G concentrations
- if you translocate faster, pulses are faster
- you can titrate to get the speed you'd like.
- translation is slowest for first couple of codons, but then speeds up. This may have to do with settling the reading frame? Much work to do here.
Ribosome is a target for antibiotics
- eg. erythromycin
- peptides exit through a channel in the 50S subunit.
- macrolide antibiotics block this channel by binding inside at narrowest point.
- They kill peptide chains at 6 bases. Are able to demonstrate this using the system.
Which model of tRNA dissociation during translation is correct
- tRNA arrival dependent model
- Translocate dependent model
Post syncrhonization of number of tRNA occupancy
- "remix our data"
- data can then be set up to synchronize an activity - eg, the 2nd binding.
Fusidic acid allows the translocation but blocks arrival of subsequent tRNA to A site.
- has no effect on departure rate of tRNA.
only ever 2 trnas at once on Ribosome. - it can happen, but not normally
Translocation dependent model is correct.
Correlating ribosome and tRNA dynamics
- towards true molecular movies
- label tRNAs... monitor fluctuation and movement
Translational processes are highly regulated
- regulation of initiation (51 and 3` UTR)
- endpoint in signallig pathways (mTOR, PKR)
- programmed changes in reading frames (frameshifts)
- control of translation mode (IRES, nromal)
- target of therapeutics (PTC124 [ribosome doesn't respect stop codons] and antibiotics)
- directly track in real time
- tRNAs dissociate from the E site post translocation and no correlation...
Paper is in Nature today.
Labels: AGBT 2010