Table 1.
A comprehensive summary of single-molecule measurements of translation dynamics in living cells
| System | Results | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Reference(s) | Cell | Protein of interest (POI) | aa | Tag | aa | Probe | RNA-tag | Probe | Initiation rate (sec) | Elongation rate (aa/sec) | Ribosome density (nt/ribo) | Expression | Mobility | Selected heterogeneity highlight |
| Repeat-epitope tags | ||||||||||||||
| Voigt et al. 2017 | U2OS | Renilla-FKBP | 418 | 4×Sun | 96 | scFV-sfGFP | 24×MS2 | NLS-MCP- Halo-JF549 | - | - | ∼380 (cytosol), ∼260–380 (ER) | 71% ± 2% | - | ER translation efficient; small fraction of cytosolic protein translated in ER |
| Gaussia | ∼180 | - | - | - | - | - | - | - | D (<0.06) ERa | |||||
| Renilla | ∼330 | - | - | - | - | |||||||||
| Pichon et al. 2016 | HeLa | Ki67 | 3177 | 56×Sun | 1344 | scFV-sfGFP (± NLS) | 24×MS2 | NLS-MCP- TagRFP-T | ∼13 | 13.2 - 18 | ∼770 | 47% B | D (0.042) | Translation efficiency is higher in translation factories than out |
| POLR2Ab | 1970 | - | - | ∼19c | 13.8 | ∼770 | 91% B | D (∼0.04) | ||||||
| DYNC1H1b | 4646 | 32×Sun | 768 | - | - | ∼480 | ∼73% B | D (0.01) 19% M (1) | ||||||
| Morisaki and McNally 2014; Morisaki et al. 2016 | U2OS | KDM5Bc | 1544 | 10×HA/ FLAG | 325 | Fab- Cy3/A488e |
24×MS2 | tdMCP- Halo-JF646 | ∼30 | 10 ± 1.3 | ∼910d | 86% ± 2% Cd | D (∼0.05) | Translation factories can be stable |
| β-actin | 374 | 12 ± 2.2 | ∼360d | 19% ± 4% Cd | D (<0.05) | |||||||||
| Histone H2B | 125 | 7.7 ± 3.2 | ∼180d | 4% ± 1% Cd | D (∼0.13) | |||||||||
| Wu et al. 2016 | U2OS | AIDc | 241 | 24×Sun | 576 | scFV-sfGFP | 24×MS2 | stdMCP-TagRFP-T/ stdMCP-stdGFP/stdMCP-Halo-JF549 | ∼45 | 5.6 ± 1.4e | ∼610 | ∼70% C | untethered:∼50% SD/ ∼50% D CytERM: ∼100% SD | β-actin 3′ UTR burst in neurons, not in U2OS; translation efficiency lower in distal dendrite |
| oxBFP-AID | 484 | 4.7 ± 0.6e | ∼640 | - | ||||||||||
| FLuc-AID | 496 | 5.0 ± 1.1e | ∼540 | - | ||||||||||
| FLuc-oxBFP-AID | 1037 | - | - | ∼690 | - | |||||||||
| Neuron | oxBFP-AID | 467 | - | - | ∼29 | 5 | - | 80% B/20% C | C in distal M to dendrite | |||||
| Yan et al. 2016 | U2OS | Kif18b | 850 | 24×Sun | 576 | scFV-sfGFP | 24×PP7 | tdPCP-3xmCherry/tdPCP-2xmCherry-CAAX | 17–43 | 3.5 ± 1.1 | 200–400 | 70% B | untethered: D (0.047) CAAX: D (1.06 × 10−3) | 5% of mRNA produce 90% of protein (Emi1 5′ UTR) |
| 10×Sun | 240 | - | 200–400 | - | ||||||||||
| 5×Sun | 120 | 3.1 ± 0.4 | 160–320 | - | ||||||||||
| BFP | 236 | 24×Sun | 576 | - | - | 4.9h | - | - | ||||||
| Wang et al. 2016 | HeLa | ODC | 473 | 24×Sun | 576 | scFV-sfGFP | 24×PP7 | tdPCP- 2xTomato/- | ∼20c | 4 | ∼260 | - | D (0.021) D (0.0053) perinuclear D (0.0013) ERi D (0.0017) ERi | Location-dependent mobility; transient burst of translation following stress (ATF4 uORFs) |
| BFP-ODC | ∼700 | - | - | - | D (0.021) | |||||||||
| MBP-ODC | ∼850 | - | - | - | - | |||||||||
| Neuron | ODC | 473 | 24xSun | 576 | scFV-sfGFP | 24xPP7 | - | - | - | - | - | D (0.006) 7% M (2.0) | ||
| TRICK | ||||||||||||||
| Halstead et al. 2015 | U2OS | NLS-3xHA-dNP799 | 368 | 6xPP7 | 405 nt | NLS-PCP- GFP | 24xMS2 | NLS-MCP- RFP | - | - | - | - | - | Oskar transcript translated posterior pole of oocyte |
| HeLa | Renilla | ∼330 | 12xPP7 | 810 nt | NLS-MCP- Halo-JF549 | - | - | - | - | - | ||||
| Oocyte | Osk | ∼600 | 6xMS2 | NLS-MCP- RFP | - | - | - | - | - | |||||
| Venus | ||||||||||||||
| Ströhl et al. 2017 | RGC | Β-actin | 374 | Venus | 239 | - | - | - | - | - | - | Synchronized C | - | Netrin-1-induced synchronized translation |
| Ifrim et al. 2015 | Neuron | PSD95 | ∼700 | Venus | 239 | - | 4×BoxB | λN22-CFP | - | - | - | - | - | PSD-95 translation dysregulated in dendrites and spines of FXS |
| Tatavarty et al. 2012 | Neuron | ARC | ∼400 | Venus | 239 | - | ∼10× UTP Cy5 | - | ∼10–15 | - | ∼200 | 12% B/88% C | - | Bursts in neuronal RNA granules |
| FMRP | ∼630 | 52% B/48% C | - | |||||||||||
| Fmr1 KO neuron | ARC | ∼400 | 24% B/76% C | - | ||||||||||
| Yu et al. 2006 | Escherichia coli | Tsr | 450 | Venus | 239 | - | - | - | - | - | - | 1.2 B per cell cycle, 4.2 molecule per B | - | Bursty translation |
TRICK, translation RNA imaging by coat protein knockoff; U2OS, human osteosarcoma; HeLa, Henrietta Lacks; RGC, retinal ganglion cell; Fmr1 KO, fragile X mental retardation 1 knockout; FKBP, FK506-binding protein; POLR2A, RNA polymerase II subunit A; DYNC1H1, cytoplasmic dynein 1 heavy chain 1; KDM5B, lysine demethylase 5B; AID, activation-induced cytidine deaminase; oxBFP, oxidizing environment variant of blue fluorescent protein; FLuc, firefly luciferase; Kif18b, kinesin family member 18b; BFP, blue fluorescent protein; ODC, ornithine decarboxylase; MBP, maltose-binding protein; NLS-3xHA-dNP799, three copies of the hemagglutinin tag fused to a nuclear localization sequence and fluorogen-activating protein; Osk, oskar maternal effect protein; PSD95, postsynaptic density protein 95; ARC, activity-regulated cytoskeleton-associated protein; FMRP, fragile X mental retardation protein; Tsr, serine chemoreceptor protein; HA, hemagglutinin; PP7, Pseudomonas phage 7; scFV, single-chain variable fragment; sfGFP, superfolder green fluorescent protein; NLS, nuclear localization sequence; Fab, antibody fragment; Cy3/A488. cyanine 3/Alexa 488; NLS-PCP-GFP, PP7 coat protein fused to a nuclear localization sequence and green fluorescent protein; MS2, bacteriophage MS2; UTP Cy 5, uridine triphosphate labeled by cyanine 5; NLS-MCP-Halo-JF549, MS2 coat protein fused to a nuclear localization sequence and a Janelia Fluor 549 HaloTag ligand; NLS-MCP-TagRFP-T, MS2 coat protein fused to a nuclear localization sequence and the variant of red fluorescent protein; tdMCP-Halo-JF646, tandem dimer MS2 coat protein fused to a Janelia Fluor 646 HaloTag ligand; stdMCP-TagRFP-T, synonymous tandem dimer MS2 coat protein fused to the variant of red fluorescent protein; tdPCP-3xmCherry, tandem dimer PP7 coat protein fused to three copies of the monomeric mCherry protein; λN22-CFP, λN22 peptide fused to cyan fluorescent protein; ER, endoplasmic reticulum; C, constitutive; B, bursty; S, subdiffusive; D, diffusive (µm2/sec); M, motored (µm/sec); UTR, untranslated region; mRNA, messenger RNA; Emi1, early mitotic inhibitor; ATF4 uORFs, activating transcription factor 4 upstream open reading frames; FXS, fragile X syndrome.
aDiffusion constant measured with RNA tag only.
bEndogenous locus.
cNot reported but calculated here from elongation rate and ribosomal density.
dPolysomes.
eMeasured with CytERM.
fEstimated from the graph.
gCalculated from the ratio.
hCodon optimized.
iTargeted to ER by coding sBFP or SMTOM upstream via P2A.