Choice of donor dye

BODIPY FL (BOF) was the donor dye in the original FRET studies (Woolhead et al., 2004, 2006). However, to reduce experimental uncertainty, we have here used NBD as the donor. εNBD-Lys is incorporated into protein at the same rate and to the same extent as unmodified Lys (Crowley et al., 1993), the spectral overlap of NBD emission and BOP absorbance is excellent (Fig. 1 B), and the instruments used here can reproducibly quantify net NBD intensities in aqueous media at ~1 nM. To directly compare NBD-BOP and BOF-BOP E values, RTCs were prepared with 90-residue nascent chains of the proteins examined in the original study, the secretory protein preprolactin (pPL) and the SSMP designated 111p (Fig. 1 C). NBD-BOP E values for the 24-residue nascent chain segments inside the ribosome tunnel were 6 ± 1% for pPLDA₉₀ and 29 ± 1% for 111pDA₉₀ (Fig. 1 D), and the corresponding BOF-BOP E values were 10 ± 1% and 47 ± 5% (Woolhead et al., 2004). Thus, both donor–acceptor pairs showed that the nascent secretory protein segment was extended within the ribosome tunnel, whereas the nascent SSMP TMS folded into a compact structure inside the tunnel.

R₀(2/3), the R₀ value assuming free rotation of the donor and acceptor dyes (κ² = 2/3), was determined experimentally to be 47 Å for the NBD-BOP pair and 57 Å for the BOF-BOP pair (Woolhead et al., 2004). Using these values and the above efficiencies, the average distance between the donor and acceptor dyes for the 111p TMS in the ribosome tunnel was found to be 55 ± 1 Å for the NBD-BOP pair and 58 ± 2 Å for the BOF-BOP pair. Because the dye separations measured by the two pairs were indistinguishable when R was near R₀ (i.e., when FRET measurements are most sensitive to changes in the distance between the donor and acceptor), the two donor–acceptor pairs are spectroscopically equivalent.

TMS2 folds in the ribosome tunnel

To determine whether the second TMS (TMS2) of a PMP folds as it moves through the tunnel, integration intermediates containing 2TM_L12DA2₁₃₀ nascent chains were prepared (Fig. 2 A; 2TM is the number of TMSs in the PMP, L12 is the size of the loop between TMS1 and TMS2, 130 is the length of the nascent chain, and DA2 indicates that the donor and acceptor dyes flank TMS2). The emission spectra obtained for the D, DA, A, and B samples are shown in Fig. 2 B, and the net D and net DA spectra are shown in Fig. 2 C. Because the presence of the acceptor decreased donor emission intensity in this experiment (E = 30%), the dyes were close enough to exhibit substantial FRET. In contrast, little FRET was detected (E = 6% in this experiment) when RTCs with 2TM_L12DA2₁₂₂ nascent chains were examined (Fig. 2 D). Because the fluorescence lifetimes and rotational rates of the donor dyes were nearly the same for 2TM_L12DA2₁₂₂ and 2TM_L12DA2₁₃₀ (Tables I and ​andII),II), the large difference in E values for the two nascent chains did not result from changes in quantum yield or a κ² effect. Instead, the ΔE can only be explained by a substantial change in donor–acceptor separation. Thus, the TMS2 segment is, on average, almost fully extended after its synthesis, but it soon folds into a more compact conformation that brings the dyes closer together inside the ribosome tunnel.

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Figure 2.

FRET-detected TMS2 folding. (A) Topogenic sequence and dye locations are depicted as in Fig. 1. TMS2 = opsin 2 (yellow). (B) Emission spectra of the 2TM_L12DA2₁₃₀ D (black), DA (red), A (purple), and B (green) samples. NBD emission peaks at 530 nm, BOP emission peaks at 596 nm, and the water Raman peak is at 556 nm. (C and D) Net normalized donor emission spectra for the D (black) and DA (red) samples of (C) 2TM_L12DA2₁₃₀ and (D) 2TM_L12DA2₁₂₂. (E and F) Average E (±SD) values obtained in three or more independent experiments for (E) 2TM_L12DA2 and (F) 2TM_L53DA2 RTCs with different nascent chain lengths and folded (black) or unfolded (gray) TMSs.

When additional lengths of 2TM_L12DA2 were examined to determine exactly when TMS2 folded, the transition from unfolded to folded was found to be surprisingly abrupt. The addition of only a single amino acid to the end of a 125-residue nascent chain resulted in a dramatic increase in E, caused by TMS2 folding into a more compact conformation when its C terminus was 7 residues from the PTC (Fig. 2 E). This sharp transition strongly suggests that TMS folding is ribosome induced and occurs at a specific location within the ribosome tunnel. The only chemical differences between the 2TM_L12DA2₁₂₅ and 2TM_L12DA2₁₂₆ samples were nascent chains that differed in length by 1 residue and the truncated mRNAs that differed in length by three nucleotides. Thus, the change in E resulted solely from a change in TMS environment within the ribosome tunnel as 2TM_L12DA2 reached a length of 126 residues. The FRET-detected conformational change in the nascent chain was presumably stabilized by ribosome–nascent chain interactions when the TMS reached that location.

Loop size does not affect folding

To determine whether the size of the TMS1–TMS2 separation had any effect on the timing of TMS2 folding, integration intermediates were prepared with nascent chains that had a longer loop between the TMSs (Fig. 2 A). The low E measured with 2TM_L53DA2₁₆₃ integration intermediates showed that extending the polypeptide by 41 residues (~140 Å if fully extended) did not cause the newly synthesized TMS2 to fold (Fig. 2 F). However, when the C terminus of TMS2 was 6 residues from the PTC, E increased sharply to 30% (Fig. 2 F). Thus, nascent chain folding inside the ribosome tunnel was independent of the length of the nascent chain, as folding occurred only after a TMS entered the ribosome tunnel and reached a certain location.

Folded TMS2 is helical

Because the two dyes are covalently attached to the peptide backbone by long flexible tethers, the FRET approach cannot determine directly the conformation of the intervening polypeptide. However, one can compare the dye separations when TMS2 is in the ribosome tunnel and when TMS2 is folded into a transmembrane α-helix in the nonpolar core of the bilayer. After 2TM_L12DA2 was fully translated and released from the translocon into the lipid bilayer, E was 31% for TMS2 with donor and acceptor dyes on opposite sides of the membrane (Fig. 2 E). This E was experimentally indistinguishable from the E values measured for TMS2 inside the tunnel when the nascent chain was 126 residues or longer, suggesting that TMS2 folds into an α-helix or an equivalently compact structure inside the ribosome tunnel.

TMS3 folds in the ribosome tunnel

TMS3, the third TMS segment in a PMP, has an orientation in the bilayer opposite to that of TMS2. Lin et al. (2011) showed that the entry of TMS3 into the tunnel triggered the reversal of the RTC structural changes elicited by TMS2. Because TMS1 and TMS2 each folded shortly after entering the tunnel, we determined whether TMS3 also folded. The E for RTCs containing 3TM_L12,18DA3₁₅₉ nascent chains (Fig. 3 A) was 5% (Fig. 3 B), so TMS3 was fully extended when newly synthesized. However, E was 37% for 3TM_L12,18DA3₁₆₇ RTCs (Fig. 3 B; dye separation was 21 residues in TMS3 and 24 in TMS2), indicating that TMS3 had folded into a compact conformation when its C terminus was 6 residues from the PTC. Thus, TMS1, TMS2, and TMS3 each folded into a compact, largely helical conformation when its C terminus was 6–7 residues from the PTC, even though the TMSs differed markedly in length, sequence, hydrophobicity, charged residues, and final bilayer orientation (Fig. S1).

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Figure 3.

FRET-detected TMS3 folding. (A) Topogenic sequence and dye locations are depicted as in Fig. 2. TMS3 = opsin 3 (magenta). (B) Average E (±SD) values obtained in three or more independent experiments for 3TM_L12,18DA3 RTCs with different nascent chain lengths and folded (black) or unfolded (gray) TMSs.

TMS folding is ribosome induced

The folding of successive TMSs at the same location in the tunnel suggested that the ribosome plays an active role in nucleating and stabilizing TMS folding. To examine this issue directly, FRET-detected folding of TMS2 inside and outside of the ribosome tunnel was compared. Parallel RNC samples with either 2TM_L12DA2₁₃₀ or 2TM_L12DA2₁₈₀ nascent chains were prepared in the absence of ER microsomes and SRP to yield RNCs with TMS2 located either inside the tunnel or in the solvent outside the tunnel (Fig. 4 A). Spectral analysis of 2TM_L12DA2₁₃₀ RNCs revealed that TMS2 was folded (E = 21%; Fig. 4 B) in the tunnel (this E with free RNCs was somewhat lower than the 29% observed with membrane-bound RNCs). In contrast, the TMS2 sequence of 2TM_L12DA2₁₈₀ was completely unfolded and extended after emerging from the tunnel (E = 4%; Fig. 4 B), despite the presence of cytosolic chaperones. Thus, the TMS2 sequence folded stably inside the aqueous (Crowley et al., 1993) ribosome tunnel, but not outside the ribosome in the aqueous medium. The ribosome therefore actively induces and stabilizes the folding of TMS2.

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Figure 4.

TMS folding is ribosome dependent. (A) The approximate locations of donor (green) and acceptor (red) dyes in 2TM_L12DA2₁₃₀ and 2TM_L12DA2₁₈₀ RTCs are shown. (B) Average E (±SD) values obtained in three or more independent experiments for these RTCs with folded (black) or unfolded (gray) TMSs.

TMS2 photocrosslinks ribosomal protein L4

To determine whether TMS2 was adjacent to ribosomal proteins inside the tunnel, integration intermediates were prepared in the dark with a photoreactive probe in TMS2 and then illuminated to initiate cross-linking to nearby molecules. By varying the distance between the PTC and the TMS2 probe, TMS proximity to ribosomal proteins could be determined at different stages of integration. Probes were incorporated into a specific site of TMS2 by adding N^ε-(5-azido-2-nitrobenzoyl)-Lys-tRNA^Lys (εANB-Lys-tRNA^Lys) to an in vitro translation of 2TM_L12K2 (K2 indicates the single Lys codon is located in TMS2). Because εANB-Lys is uncharged, TMS hydrophobicity was retained. To examine TMS2 environment at higher resolution, probes were placed at four adjacent TMS2 sites (1 site/sample; Fig. S1). This approach allowed us to determine whether any observed proximity was random or specific because the probes would project from different sides of a folded TMS helix (McCormick et al., 2003).

When four different probe sites (106–109; Fig. S1) were examined in parallel at each of four different lengths (122, 125, 126, 130) of ³⁵S-labeled nascent 2TM_L12K2, relatively few high molecular mass radioactive bands were observed in SDS gels of total sample proteins from either membrane-bound RTCs (Fig. 5 A) or free RNCs (Fig. 5 B). Because the high molecular mass bands were absent in samples that lacked the ANB probe or were not illuminated, these bands resulted from nascent chain photocrosslinking to proteins inside the ribosome tunnel. The primary photocrosslinking target for all probe sites and nascent chain lengths for both RTCs and RNCs was a 40-kD protein, most likely ribosomal protein L4. No antibodies are available to confirm this identification by immunoprecipitation, but L4 has a molecular mass near 40 kD and is the largest protein exposed inside the 60S tunnel (Armache et al., 2010b; Ben-Shem et al., 2010).

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Figure 5.

TMS2 photocrosslinking to ribosomal proteins. Membrane-bound (A) or free (B) RNCs containing nascent ³⁵S-labeled 2TM_L12K2 chains of different lengths were prepared with a photoreactive probe at one of four different TMS2 sites (106–109; Fig. S1), photolyzed, and analyzed by SDS-PAGE. Photoadducts containing L4, L17, and L39 are identified by , •, and , respectively. (C) Free RNCs containing a ³⁵S-labeled 2TM_L12K2₁₂₆ nascent chain with a probe at either 106 or 107 were photolyzed and analyzed (lanes 1 and 2 = total proteins; lanes 3 and 4 = proteins precipitated with antibodies specific for L17).

Interestingly, the extent of TMS2 cross-linking to L4 was the same from all four probe sites, irrespective of the length of the nascent chain. Because L4 does not circumscribe the ribosome tunnel and instead is exposed only on one side, the fact that L4 was photocrosslinked equally well from each of the four different probe locations in TMS2 (Fig. 5, A and B) reveals that nascent chains in the tunnel were oriented randomly relative to L4. Moreover, the insensitivity of photoadduct formation to nascent chain length indicates that similar numbers of TMS2 probes were adjacent to L4 during photolysis of nascent chains 122–130 residues long. Because the number of TMS2 probes reacting with L4 did not change as the nascent chain was lengthened by 8 residues, it appears that the extent of dynamic back and forth motion of individual nascent chains in the ribosome tunnel was sufficient to equalize the average probe concentration next to L4. Thus, the nascent chains that react covalently with L4 are moving and rotating freely in the ribosome tunnel, and are not bound to its surface. TMS2 photocrosslinking to L4 in the tunnel therefore results from a random and dynamically constant spatial proximity over this range of nascent chain lengths, not from TMS2-L4 association.

TMS2 is adjacent to L17 and L39

TMS2 also photocrosslinked to smaller proteins with masses near 18 or 7 kD (Fig. 5, A and B), most likely L17 (18 kD) and L39 (7 kD) because these are the only proteins besides L4 exposed inside the eukaryotic ribosome tunnel (Armache et al., 2010b; Ben-Shem et al., 2010). Yet the electrophoretic mobilities of the two smaller photoadduct bands in lanes 9 and 13 are distinctly different from those in lanes 10 and 14 (Fig. 5, A and B), raising the possibility that probes in different TMS2 positions cross-linked to different proteins. To ascertain whether either or both of the photoadduct bands near 30 kD in lanes 9 and 10 of Fig. 5 A contained L17, samples were photolyzed and immunoprecipitated with antibodies specific for L17. The results shown in Fig. 5 C reveal that the 106 and 107 photoadducts each contained both L17 and the radioactive nascent chain. Thus, TMS2 cross-links to L17 from both the 106 and 107 probe sites.

No antibodies are available to immunoprecipitate L39, but the probes at 106 and 107 also appear to react covalently with L39 (Fig. 5 A).

TMS2 folding coincides with its exposure to L17

The extent of TMS2-L17 photocrosslinking varied with nascent chain length because the ~30-kD photoadduct yield was close to zero with 122mers, maximal with 126mers and 130mers, and 22% of maximal for 125mers; the yields of putative TMS2-L39 photoadduct varied similarly (Fig. 5, A and B). The sharp increase in L17 and L39 photocrosslinking as the nascent chain lengthened by a single residue is striking, especially given the insensitivity of L4 photocrosslinking to nascent chain length between 122 and 130. Equally striking was the folding of TMS2 into a helical conformation when 2TM_L12K2 reached a length of 126 residues (Fig. 2 E). The very strong correlation between the changes in TMS2 conformation and environment strongly suggests that TMS folding and TMS accessibility to L17 are linked, and that only folded TMSs are adjacent to L17. Furthermore, because the nascent chain length dependence of TMS2-L17 photocrosslinking was the same for both membrane-bound RTCs (Fig. 5 A) and free RNCs (Fig. 5 B), ribosome-induced TMS folding and access to L17 is an intrinsic property of all ribosomes, not just membrane-bound ribosomes.

Helical TMS2 binds to L17 in a specific orientation

L4 was randomly and uniformly cross-linked from four different probe sites in TMS2 (Fig. 5 A), but TMS2 photocrosslinking to L17 was very specific and nonrandom when TMS2 was folded into a helical conformation in 126- and 130-residue nascent chains. Probes at 106 and 107 both cross-linked efficiently to L17, whereas very little cross-linking was observed with probes at 108 and 109 (Fig. 5, A and C). This result suggests that the helical surfaces from which the 106 and 107 probes projected were facing L17, whereas the probes at 108 and 109 projected into the aqueous ribosome tunnel or reacted with rRNA (cross-linking to rRNA was not examined in this study).

The different electrophoretic mobilities of the 106 and 107 photoadducts (Fig. 5 C) are best explained by the probes at 106 and 107 reacting with two different sites on L17 (Plath et al., 1998). Furthermore, because lanes 9 and 13 contained only a single radioactive 30-kD band (Fig. 5 A), the 106 probe was always located in the same position relative to L17, and never reacted with L17 near where the 107 probe was always located. It therefore appears that the adjacent TMS2 106 and 107 probes/residues in the TMS2 helix each projected toward distinct, nonoverlapping regions of L17. The only mechanism by which TMS2 and its probes could stably and reproducibly occupy the same position in the ribosome tunnel adjacent to L17 is if TMS2 were bound to specific ribosomal components and held in a fixed orientation (McCormick et al., 2003). The specificity of L17 cross-linking to TMS2 therefore strongly indicates that helical TMS2 binds to the tunnel surface adjacent to L17.

Dynamic TMS location and conformation

L4, L17, and L39 are exposed to the aqueous nascent chain pathway at different locations inside the ribosome tunnel (Armache et al., 2010b; Ben-Shem et al., 2010), yet 2TM_L12K2₁₂₆ and 2TM_L12K2₁₃₀ nascent chains with probes at either 106 or 107 in TMS2 form photoadducts with L4, L17, and L39 in the same sample (Fig. 5, A and B). Because each nascent chain has only a single probe, each sample contains TMS2 sequences that are adjacent to each target protein at the time of photolysis. For TMS2s to be positioned at different ribosome tunnel locations at the same time, the nascent chains must be dynamically diffusing back and forth in the ribosome tunnel. However, the distribution of TMS2s inside the tunnel cannot be determined from the relative band intensities because photoadduct yield is critically dependent on the unknown efficiency of probe reaction with exposed ribosomal protein residue(s) and the extent of their exposure to the tunnel.

TMS2 conformation also varies dynamically inside the ribosome tunnel. TMS2 photocrosslinked to L4 while unfolded and oriented randomly, whereas L17 was photocrosslinked when folded TMS2 is bound to the tunnel wall in a specific orientation. Thus, each RTC sample contained folded and unfolded TMS2 sequences. The apparently facile transition between helical and unfolded TMS2 conformational states does not conflict with the FRET data because the E values are averages. The measured E values therefore reflect the predominant, but not the only, conformation at a given nascent chain length (Fig. 2 E).

Folded TMS3 photocrosslinks L17

When photoreactive probes were positioned at each of four TMS3 sites in 3TM_L12,18K3 nascent chains of different lengths (one site/sample; Fig. S1), the photocrosslinking pattern was very similar to that observed with TMS2 probes: L4 was cross-linked equally from each of the four TMS3 sites, indicating that those nascent chains were rotating randomly and freely in the tunnel when illuminated; photoadducts containing L17 and L39 were detected only after the TMS3 folded (i.e., in nascent chains 163 residues or longer); and L17 photocrosslinking was asymmetric because probes at 145 and 146 cross-linked more efficiently to L17 than probes at 144 and 147 (Fig. 6). Thus, folded TMS3 appears to be adjacent to and photocrosslink L17 and L39 after binding to the ribosome tunnel surface in a specific orientation.

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Figure 6.

TMS3 proximity to ribosomal proteins. Free RNCs containing nascent ³⁵S-labeled 3TM_L12,18K3 chains of different lengths were prepared with a photoreactive probe at one of four different TMS3 sites (144–147; Fig. S1) and photolyzed. Photoadducts are identified as in Fig. 5.

Interestingly, the two TMS3-L17 photoadducts did not differ significantly in electrophoretic mobility. Hence, it appears that TMS2 and TMS3 do not bind adjacent to L17 in the same way, a result that is not surprising given their different sequences and hence binding surfaces.

TMS folding correlates with RTC changes

Three experimentally detectable events occurred when the 2TM_L12 nascent chain reached a length of 126 residues: TMS2 folded into a helix (Fig. 2 E), TMS2 bound to the ribosome tunnel surface adjacent to L17 (Fig. 5 A), and TMS2 exposure switched from cytosolic to lumenal (Lin et al., 2011), as depicted in Fig. 7. The coincidence of these events was not accidental because changing the loop size did not uncouple these three events (Fig. 2 F; Lin et al., 2011). TMS3 triggered similar events when a 3TM_L12,18 nascent chain reached a length of 163 residues: TMS3 folded into a helix (Fig. 3 B), TMS3 bound to L17 (Fig. 6), and TMS3 exposure changed from lumenal to cytosolic (Lin et al., 2011). TMS1 movement into the ribosomal tunnel and folding elicited the same changes as did TMS3 (Liao et al., 1997; Woolhead et al., 2004). Although these three events, TMS folding, TMS binding to L17, and an inversion of TMS exposure to cytosol or lumen, were temporally coincident for each of three successive TMSs, they were not spatially coincident: TMS folding and binding to L17 occurred at the constriction far inside the ribosome tunnel, while the lumenal end of the aqueous translocon pore was alternately closed or opened on the opposite side of the ER membrane.

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Figure 7.

L17 links TMS folding with changes in the RTC. TMS2 folding coincides with its binding to L17 (red) and also with a change in nascent chain exposure from cytosolic to lumenal, strongly suggesting that L17, which extends from the constriction to the ribosome surface near the translocon (T, yellow), acts as a conduit for communicating the imminent arrival of a TMS at the translocon. Although this cartoon depicts the ribosome tunnel as fully sealed off from the cytosol when TMS2 is still far inside the tunnel, this is unlikely to be the case in vivo because ongoing translation will move TMS2 down the tunnel as the RTC and other molecules are assembled and rearranged at the membrane. Thus, TMS2 is likely to be in or near the translocon by the time the depicted changes are completed. Cytosolic access to the ribosome tunnel is blocked either by a conformational change in the RTC and/or by an unknown protein (?, black), here shown binding to the RTC to block tunnel access. BiP-mediated pore closure is reversed by ATP-dependent release of BiP from an unidentified J domain–containing ER membrane protein (J, green; Alder et al., 2005). RAMP4 (R, dark blue) is adjacent to L17 when the nascent chain is exposed to the cytosol, whereas Sec61β (β, magenta) is adjacent to the nascent chain throughout (Pool, 2009).

To coordinate the timing of events separated by more than 110Å, a molecular linkage that extends from the tunnel constriction through the ribosome and membrane to the ER lumen must exist and act as a signal transduction pathway. Furthermore, because these structural changes occur only after the C-terminal end of a nascent chain TMS has moved 6–7 residues into the ribosome tunnel and folded, the data indicate that nascent chain folding initiates and thereby regulates what happens at each end of the translocon pore.

RTCs and RNCs for fluorescence experiments

In vitro translations (500 µl) to prepare RNCs contained 20 mM Hepes, pH 7.5; 2.8–3.5 mM (optimal concentration was determined experimentally for each lot and combination of macromolecular components) Mg(OAc)₂; 100–130 mM (optimized) KOAc, pH 7.5; 1 mM DTT; 0.2 mM spermidine; 8 µM S-adenosyl-methionine; 1x protease inhibitors (Erickson and Blobel, 1983); 0.2 U/µl RNasin (Promega); 40 µl of an energy-generating system containing 375 µM of each of the 20 amino acids except lysine, 120 mM creatine phosphate, and 0.12 U/µl\e creatine phosphokinase; 60–80 µl (optimized) wheat germ extract (Erickson and Blobel, 1983); 40 µl mRNA; and 300 pmol of each of two modified aa-tRNAs as detailed below (tRNA Probes, LLC). When RTCs (membrane-bound RNCs) were desired, 40 nM purified canine SRP and 80 eqs of canine salt-washed ER rough microsomes (tRNA Probes, LLC) were also added to the above translations. mRNAs were incubated at 65°C for 5 min and then quickly cooled on ice before addition to translations to minimize any inhibition by mRNA secondary structure. Before addition of mRNA and tRNA, translations were incubated at 26°C for 7 min to complete the translation of any residual endogenous mRNA fragments. After mRNA and tRNA addition, reactions were incubated at 26°C for another 30–35 min. When working with longer nascent chain lengths (171 amino acid residues and longer), the aa-tRNAs were added 5 min after the beginning of translation to reduce aa-tRNA deacylation before incorporation.

Four samples were always prepared in parallel that differed only in the identities of the modified aa-tRNAs added to a translation: D received εNBD-[¹⁴C]Lys-tRNA^Lys and εAc-[³H]Lys-tRNA^amb; DA received εNBD-[¹⁴C]Lys-tRNA^Lys and εBOP-[³H]Lys-tRNA^amb; A received εAc-[¹⁴C]Lys-tRNA^Lys and εBOP-[³H]Lys-tRNA^amb; and B received εAc-[¹⁴C]Lys-tRNA^Lys and εAc-[³H]Lys-tRNA^amb. RTCs were purified away from NBD not in RTCs by a high salt wash and then gel filtration. At the end of a translation, each sample was adjusted to 500 mM in KOAc and incubated on ice for 10 min. Each sample was then loaded onto a separate gel filtration column (Sepharose CL-2B, 0.7 cm I.D. x 50 cm; column resin must be replaced every 3–4 runs to avoid sample contamination by materials that adsorb to the resin) that had been preequilibrated in buffer A (50 mM Hepes, pH 7.5, 40 mM KOAc, pH 7.5, 5 mM Mg(OAc)₂) and then preloaded with 2 ml of buffer B (20 mM Hepes, pH 7.5, 500 mM KOAc, pH 7.5, 3.2 mM Mg(OAc)₂; Haigh and Johnson, 2002). After chromatography at a very slow rate (2–3 drops/min; 4°C) to ensure dissociation of noncovalently bound NBDs, the membrane-bound RNCs (= RTCs) eluted in the void volume, typically in 1.1 ml (two 550 µl fractions). Free RNC samples lacking microsomes were treated the same way, except that no high-salt wash was done and Sepharose CL-6B (1.5 cm I.D. x 20 cm) was used instead of Sepharose CL-2B (microsomes elute in the void volume of Sepharose CL-2B columns, while free RNCs elute in the void volume of Sepharose CL-6B columns). After gel filtration, the light-scattering signals of the four parallel samples were measured using either λ_ex = 405 nm, λ_em = 420 nm, or λ_ex = 468 nm, λ_em = 485 nm (no significant difference was observed between these choices) and equalized by diluting the samples as necessary before initiating spectral measurements.

More than 50% of the εNBD-Lys-tRNA^Lys added to a wheat germ translation of preprolactin incorporated its amino acid into protein (Crowley et al., 1993). However, in the PMP FRET studies, only ~0.5% of the εNBD-Lys added to the translation was recovered in the void-volume gel filtration fractions that contained the 2TM and 3TM nascent chains in microsome-bound RTCs due to a combination of effects: losses during purification; less efficient translation of the PMPs than of pPL; insertion of the εNBD-Lys late in the nascent PMP sequence; and the requirement for εBOP-Lys-tRNA^amb to translate the amber stop codon instead of a termination factor. The latter two effects increased the loss of εNBD-Lys-tRNA^Lys due to deacylation, especially because the large size of the BOP dye slowed its rate of incorporation. The NBD concentration in the samples analyzed by FRET was 1–2 nM, whereas the acceptor concentration was three- to fourfold larger (residual Lys-tRNA^Lys competition with εNBD-Lys-tRNA^Lys reduced the amount of NBD incorporated in nascent chains containing εBOP-Lys; Krieg et al., 1989).

Steady-state fluorescence

Steady-state measurements were made at 4°C in buffer A on a Spex Fluorolog 3–22 or SLM-8100 photon-counting spectrofluorimeter with a 450-W xenon lamp, two excitation monochromators, one (SLM) or two (Spex) emission monochromators, and a cooled low-background Hamamatsu R928 PMT. Samples were maintained at 4°C while nitrogen was flushed through the sample compartment to prevent condensation from forming on the 4 × 4-mm quartz microcuvettes. After additions to a sample, the solution was mixed thoroughly with a 2 × 2-mm magnetic stirring bar as described previously (Dell et al., 1990). Samples were then placed in the sample chamber for 5 min and allowed to equilibrate to 4°C before any measurements were made. To obtain an emission intensity measurement, five successive 5-second integrations of emission intensity were recorded and averaged.

FRET

D, DA, A, and B samples that differ only in the number of fluorescent dyes (0, 1, or 2) in the nascent chain were prepared, purified, and examined in parallel to correct for background spectral signals. Aliquots (250 µl) of the purified and scattering-equalized D, DA, A, and B samples of RTCs or RNCs were placed into each of four cuvettes. The emission intensities (F) were measured at λ_ex = 468 nm, λ_em = 530 nm (4 nm bandpass) to focus on the donor dye emission. Because the signal recorded for the blank is due to light scattering and background fluorescence, and the signal from A is caused by light scattering, background fluorescence, and direct excitation of the acceptor (i.e., not emission due to FRET from the donor dye), the net emission intensities due to donor dye fluorescence in the D and DA samples, net F_D and net F_DA, were determined by subtracting F_B from F_D and F_A from F_DA.

To accurately compare net F_DA with net F_D, the number of donor dyes in the D and DA samples had to be determined by quantifying the εNBD-[¹⁴C]Lys content in 200 µl of each sample (Crowley et al., 1993). The double-label counting efficiencies of the liquid scintillation counter were determined experimentally, using standard solutions of [³H]H₂O and [¹⁴C]glucose in the same scintillation cocktail system used for the samples, to be 0.60 and 0.16 for ¹⁴C, and 0.0 and 0.4 for ³H in the two windows. F_D was then normalized to the same number of donor dyes as in F_DA. Because the NBD is too far from BOP to affect its absorbance, the FRET efficiency E is given by E = 1 – (Q_DA/Q_D) = 1 – (normalized net F_DA)/(normalized net F_D). E quantifies the acceptor-dependent quenching of donor emission intensity due to FRET. Although the average distance between the donor and acceptor dyes, R, can be calculated from E, the uncertainties associated with κ², the lengths of the probe-to-nascent chain tethers, and the dynamic equilibrium between nascent chain conformations (see Results) preclude a definitive determination of the actual polypeptide conformation. Thus, to monitor nascent chain folding, we have focused on changes in E, and hence R, as the nascent chain moves through the ribosomal tunnel.

Determination of R₀

R₀, the distance between the donor and acceptor dyes at which FRET efficiency is 50%, was determined experimentally (Johnson et al., 1982) for the NBD-BOP donor-acceptor pair in RTC complexes. R₀⁶ = (8.79 × 10⁻⁵)QJ_DAn⁻⁴κ², where R₀ is in Å, Q is the quantum yield of the donor in the absence of the acceptor, J_DA is the spectral overlap integral in M⁻¹cm⁻¹nm⁴, n is the refractive index of the medium between donor and acceptor, and κ² is a geometric factor that depends on the relative orientation of the transition dipoles of the donor and acceptor dyes. The overlap of the corrected NBD emission spectrum and the BOP (ε_{575 nm} = 83,000 M⁻¹cm⁻¹) absorbance spectrum in 2TM_L12DA2 RTCs (Fig. 2 A) yielded a value of 4.7 × 10¹⁵ M⁻¹cm⁻¹nm⁴ for J_DA, and n was assumed to be 1.4 (Johnson et al., 1982).

The quantum yield of NBD in free εNBD-Lys was determined experimentally (Mutucumarana et al., 1992) using disodium fluorescein in 0.1 M NaOH as the standard (Q = 0.92; Weber and Teale, 1957). The corrected emission intensities (λ_ex = 468 nm) were integrated from 500 to 625 nm, and the Q of εNBD-Lys was found to be 0.04 in buffer A. The fluorescence lifetime of εNBD-Lys was 1.3 ns in buffer A, but the average fluorescence lifetime, <τ>, was much higher when the NBD was incorporated in or next to a nascent chain TMS and located in the ribosome tunnel (Table I; Lin et al., 2011). If one assumes that the NBD extinction coefficient is not significantly different for free εNBD-Lys and εNBD-Lys in an RTC-bound nascent chain, then Q is directly proportional to τ. The average quantum yield of NBD in the ribosome tunnel is given by Q_NBD-RTC/Q_NBD-free = τ_NBD-RTC/τ_NBD-free = Q_NBD-RTC/0.04 = 4.7–5.0/1.3, and Q_NBD-RTC = 0.15.

Because κ² cannot be determined experimentally in a nonrigid system, R₀ is typically calculated by assuming that the orientations of the donor and acceptor transition dipoles are dynamically randomized during the excited-state lifetime of the donor dye. For such randomly oriented dyes, κ² = 2/3, and the resulting R₀ value is designated R₀(2/3). Values for R calculated assuming κ² = 2/3 typically differ by less than 10% from those determined by crystallography when such comparisons can be made (e.g., Stryer, 1978; Wu and Brand, 1992; also compare tRNA-tRNA distance in solution by FRET [Johnson et al., 1982] and in a crystal structure [Yusupov et al., 2001]). The measured R₀(2/3) for the NBD-BOP in the membrane-bound PMP RNCs was therefore 47 Å.

k² Uncertainty

The theoretical upper and lower limits of κ², and hence of R₀, can be determined from anisotropy (r) or polarization data that indicate the freedom of rotation of the dyes in the sample. The measured anisotropy values for the NBD and BOP dyes were the same for probes flanking TMS1, TMS2, and TMS3, and for all lengths of nascent chains in membrane-bound RNCs (Table II; Woolhead et al., 2004). Furthermore, the same anisotropies were observed after the full-length protein had been translated and released into the membrane bilayer. Because the anisotropy averaged 0.29 for NBD and 0.29 for BOP, each dye had significant freedom of rotation in our samples, and was not bound tightly to the ribosome. Furthermore, the similarity in the anisotropies inside and outside the ribosome tunnel indicate that the rotation of the large BOP dye was not detectably restricted by the spatial limitations of the eukaryotic ribosome tunnel, perhaps because the BOP is located at the end of a flexible lysine side chain. We observed the same phenomenon with BOF and BOP dyes flanking TMS1 (Woolhead et al., 2004).

The 0.29 anisotropies yield a maximum range of 0.10 to 3.25 for κ² in our samples (Dale et al., 1979). This corresponds to a −27% to +30% maximum uncertainty in R₀ due to orientation effects. However, as discussed previously (Johnson et al., 1982; Watson et al., 1995; Woolhead et al., 2004), the actual uncertainty is closer to 10%, largely because of the statistical improbability of some dye orientations (Hillel and Wu, 1976; Stryer, 1978; Wu and Brand, 1992) and the flexibility of the dye tethers (Wu and Brand, 1992).

Time-resolved fluorescence

Fluorescence lifetimes were measured as detailed in Lin et al. (2011). The background phase and modulation data from a sample lacking NBD were subtracted from the corresponding data from an equivalent sample containing NBD that was prepared in parallel (Reinhart et al., 1991). Background-subtracted data from three or more independent experiments were combined and fit to several different models to determine which model provided the simplest fit while still yielding a low χ² value using Vinci multidimensional fluorescence spectroscopy analysis software (ISS, Inc.). The best fit was almost always obtained by assuming two discrete exponential decay components. The fit of the data were not significantly improved by assuming the samples contained three components with distinguishable lifetimes, nor by using a Lorentzian fit instead of a discrete fit. The molar fraction of dyes with τ_n is given by f_n, from which the average lifetime, <τ>, was calculated.

To examine the ribosome dependency of the observed lifetime, the NBD τ was measured for free 2TM_L12DA2₁₃₀ RNCs in the absence of the acceptor dyes (Table I). The free RNCs were then treated with 2 mM puromycin (37°C, 30 min), followed by 5 mM EDTA (pH 7.5) and 20 µg/ml RNase A (final concentrations) at 37°C for 30 min to release the nascent chains from ribosomes. After measuring the NBD τ of this sample, proteinase K was added to 0.1 mg/ml and the sample was incubated at 37°C for 30 min to digest the nascent chain before measuring the NBD τ again.

Photocrosslinking experiments

In vitro translations (50 µl) contained the above components with 30 pmol of εANB-[¹⁴C]Lys-tRNA^Lys (tRNA Probes, LLC), 50 µCi of [³⁵S]Met, an excess of the desired length of truncated mRNA containing a single Lys codon at the desired position, and glutathione instead of DTT. For RTCs, 40 nM SRP and 16 equivalents of column-washed microsomes (both from tRNA Probes, LLC) were also added. Samples were incubated at 26°C for 30 min in the dark and then photolyzed as before (McCormick et al., 2003). After photolysis, free RNCs or membrane-bound RTCs were centrifuged through a sucrose cushion buffer (130 µl; 0.5 M sucrose, 25 mM Hepes, pH 7.5, 120 mM KOAc, pH 7.5, 3 mM Mg(OAc)₂) in a TLA-100 rotor (Beckman Coulter; 100,000 rpm; 4°C; 3 min for RTCs or 10 min for free RNCs). Pellets destined for immunoprecipitation are discussed below; all other samples were resuspended in 50 µl of 50 mM Hepes, 5 mM EDTA at pH 7.5 and incubated with 1 µg of RNase A (26°C, 10 min) to remove residual peptidyl-tRNA before the addition of sample buffer and analysis by 10–15% SDS-PAGE as described previously (McCormick et al., 2003). Radioactive bands were visualized using a phosphorimager (model FX; Bio-Rad Laboratories).

Immunoprecipitations

Pellets of photolyzed samples were resuspended in 50 µl of 3% (wt/vol) SDS, 50 mM Tris-HCl, pH 7.5, and incubated (55°C, 30 min) before the volume was adjusted to 500 µl with buffer B (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% [vol/vol] Triton X-100). The diluted samples were precleared by rocking with 30 µl of protein A–Sepharose at room temperature for 1 h before the Sepharose beads were removed by sedimentation. 4 μl of rabbit antiserum against the N terminus of L17 (Pool, 2009) were added to each supernatant, and the samples were rocked overnight at 4°C. After the addition of 40 µl of protein A–Sepharose, each sample was rocked for another 2 h at 4°C. The immunoprecipitated material was recovered by sedimentation, washed twice with buffer B, and once with buffer B lacking Triton X-100. After RNase treatment and SDS-PAGE as above, the radioactive immunoprecipitated material was visualized using a phosphorimager (model FX; Bio-Rad Laboratories).

We thank Yiwei Miao and Yuanlong Shao for technical assistance.

Support was provided by National Institutes of Health grant GM26494 (A.E. Johnson), National Science Foundation grant EF-0623664 (A.E. Johnson), Robert A. Welch Foundation Chair grant BE-0017 (A.E. Johnson), and BBSRC grant BB/H007202 (M.R. Pool).

A.E. Johnson is a founder of tRNA Probes, LLC.