>AGBT 2010 – Joseph Puglisi – Stanford University School of Meicine

>The Molecular Choreography of Translation

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

Translation
– 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

Ribosome
– 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.]

Basic questions:
= timing of factor
– initiation pathway
– origins of translational fidelity
– mechanisms

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.

Solution:
– 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)

Summary:
– directly track in real time
– tRNAs dissociate from the E site post translocation and no correlation…

Paper is in Nature today.

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