Ribosomes, translation in laser trap

Following translation by single ribosomes one codonat a time. Wen et al Nature 452, 598 (2008)

Note Nobel this year for ribosome structureimportance of structural work cited in intro to this paper!

Cartoons from http://cellbio.utmb.edu/CELLBIO/ribosome.htm

Translation performed by “ribosomes”

~ 20 nm

30S subunit 1540nt rna 21 proteins

50S subunit 120nt rna 2900nt rna 34 proteins

If you could watchthis step by step,what might be theslowest (“rate-limiting”) steps?How many wouldthere be? Howwould the ribosomemove in time?

Experimental system

mRNA made in vitroby T7 RNA pol fromDNA templatesdesigned to producedifferent “hairpins”

Stall ribosome by withholding particulartRNA; then add it and“start the clock”

record F vs xas function of t

x

d = F/k

Mixedaa’s

MostlyV, E

Alt. runsof V & E

Evidence that ribosome can read through ds stem (Fig. S3)

1, ribosome stalled at base of stem (F11);it takes 23pN to meltstem ( )

2a, same as 1 but pull only to 20pN

then, 2b add missing aa-tRNA, stem gradually melts due toribosome reading through stem, melting it as it goes

After some time, increase force: 2c shows no sudden increase in extension, i.e. no evidence of residual stem

Main result – at constantforce, extension proceeds in steps – aresteps convincing?

Histogram of distancesbetween all pairs of data points shows steps clearly;steps ~2.7nm (precision!)

WLC model predicts2.7nm @20pN <->6 bases=>melting 3 bases at a timeCould you see 1.35nm step?

“Translocation”really = unwinding;translocation couldoccur during subsequent pause

Expanded time course of step

“The most parsimonious interpretation … is that unwindingand translocation … occur simultaneously…”

What (besides step height) can one analyze in such traces?

Assume rate limitingevents occur at randomat rate k (per sec)

p(1st event at time t)dt= k e-kt dt p-> k as t-> 0

Suppose 2 random, sequential events required, rates k1, k2

p(2nd event at time t)dt = p1st(t’)dt’ p2nd(t-t’) dt integrated over t’ from 0 to t. Find p(t) = [k1k2/(k2-k1)](e-k1t – e-k2t)dtFor k1=k2, p = k2te-ktnote p-> 0 as t-> 0“Translocation” time dn best fit by 3 sequential events Does this identify what the events are? Bp melting?

With longer hairpin, pause times (extension rates)are quite variable

different colors = identical RNAs

where are pauses occuring?

why?

Looking for correlations between sequence and pause sites

Some clustering of pause sites (blue) downstream of “SD”AGGAGG. Complementary seq exists in 16S ribosomal RNA, previously implicated in ribosome binding to mRNA. Pauses eliminated by mutating to AAG, but this would also change binding to tRNA; is the D pause rate statistically significant?

Pauses can beterminated bypulling on the rna(promotes un-winding)

Dwell times also notfit by single exponentialbut this data lessreliable: only first binhas too few occurrences,and only shortest dwelltimes were analyzed – long dwell times were

attributed to unknown “other” phenomena.

Conclusions

Single ribosome study allows detection of steps & pausesthat can’t be studied in ensemble (bulk) studiesbecause you can’t synchronize multiple ribosomes

Not clear if steps are due to ribosomal translocation orribosomal melting of 2ary structure in template

If shorter distances can be reliably resolved (~ 1nm!), would be better to observe ribosome on less structured RNA template

Earlier Bustamante paper current paperWhy does 5pN pull to ~ 90% of L on left but <10% on right?

Questions …

extension

Fig S1a – said to show that translation melts stem sincestalling at sites further along stem results in smallerextensions at 23pN. Why are curves sequentially dis-placed to right? Rips at lower F (arrow heads) said to be due to 2ary structure 5’ of ribosome. Why are these variable?

Fig S1B. Pulling curves after various times of translation.Why does rip size decrease? Why do pre-rip portions of curves differ? What would you predict if structures were relaxed and re-pulled after ribosome had read through?

Fig. S4 VE274hp a:1, stall at 1st E, pull and relax; 2a pullto 17pn, hold at constant position, 2b add E-tRNA; 2cpull again. Why does F decrease (2b)? Why does extensionincrease as F decreases (panel b)?

Appreciate precision – detecting ~nm displacements in liquid, despite Brownian motion (damped to some extent by tightness of laser trap spring)

They see 3 base step, hint of 1 base substeps

Extract interesting observations even from “artifacts” e.g. peculiarly long pauses

? Shine Delgarno seq related pauses, perhapsaccidentally found because E encoded by GAN ? “clogging” of protein exit site in ribsosomeby hydrophobic poly Val

Preview of next week’s topicMicrotubules (from Howard & Hyman, Nature 422:753 (03)

Part of cell “cytoskeleton”

fibers often radiate outfrom site near nucleuscalled centrosome

cell presumably stainedw/ fluorescent anti-tubulin

Microtubules

Made of 2 proteins , tubulin a and , b each ~ 500 aamonomers -> -a b dimer (8nm long), dimers -> - - ... ab ab ab protofilamentsprotofilaments -> tubes (microtubule) ~13 protofilaments/tube circumferencetube diameter ~25nm, length up to 25 mm (1000:1!)persistence length ~250mm; like Reif DNA tubes!

Tube is polarized: a at one end b at the othergrow in direction of + end

Tube growth and disassembly controlled by GTP binding

b tubulin can bind GTP and “hydrolyze” it to GDP + PIn GTP-bound state, conform. favors dimer binding to + endThis increases rate of GTP hydrolysis in adjacent dimerInternal dimers in GDP state prefer curved conformation but

are held straight by binding to adjacent dimers in tubeIf end units hydrolyze GTP -> end dimers rapidly dissociate

-> stochasticgrowth &retraction =dynamicinstability

Label tubulin with green fluorescent protein in vivo ->Tubules continually grow, retract (“catastrophe”),

regrow (“rescue”)

“Dynamic instability”

http://www.youtube.com/watch?v=ZL3_BwrB6AMhttp://www.youtube.com/watch?v=E1XczyCkN20&NR=1

GFP-label protein that binds microtubule +endshttp://www.youtube.com/watch?v=9ICqcZ99qGk

Ends of tubules often associated with structures that get moved around inside cells by growth/retraction of tubules

E.g. centrosome divideschromosomes duplicate + ends of tubules attach to chromosomes “catastrophe” pulls chromosome copies to

what will become daughter cells

“Kinesin” proteins bind microtubules and “walk” alongthem, hydrolyzing ATP as they move

Some kinesins have 2 heads, which alternately bindand release tubulin, “bipedal” walkers (recall DNA)

What is role of ATP hydrolysis?

http://www.youtube.com/watch?v=lLxlBB9ZBj4

If you immobilize kinesins on surface, addfluorescently labeled microtubules + ATP ->microtubules move along immobile “motors”

http://www.youtube.com/watch?v=hqIRNekAdfA

shows motor system only requires motor protein + ATP+ track (tubulin)