Interaction of Nascent Chains with the Ribosomal Tunnel Proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae *

Background: Rpl4, Rpl17, and Rpl39 expose patch-like domains to the empty ribosomal polypeptide tunnel. Results: Rpl4 contacts various nascent chain residues, even far from its patch-like domain. Rpl17 contacts only hydrophobic α-helical segments, other nearby nascent chain segments are excluded. Conclusion: The tunnel topology of Rpl4 and Rpl17 must be flexible. Significance: The amino acid sequence of nascent chains affects the topology of Rpl4 and Rpl17. As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His6-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.

Newly synthesized polypeptides exit the ribosome through a tunnel of ϳ100 Å. The exit tunnel is conserved and structural details of the ribosomal tunnels from all kingdoms of life are available (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In contrast to other polypeptide-conducting assemblies, for example the translocon in the endoplasmic reticulum membrane, the ribosomal tunnel is not exclusively made of proteins but built of ribosomal RNA with intermingled domains of only three ribosomal proteins (see Fig. 1, A and B). The tunnel originates at the peptidyl transferase center (PTC), 2 traverses the body of the large subunit, and exits at a platform on the opposite side. Near the PTC the tunnel wall consists of ribosomal RNA only; at a distance of ϳ20 Å, the tunnel bends and its diameter narrows to ϳ10 Å. Adjacent to this most narrow constriction, two universally conserved ribosomal proteins contribute to the tunnel wall (see Fig. 1B). Rpl4 (L4 in archaea and eubacteria) exposes a patch-like domain to the lumen (see Fig. 1C), Rpl17 (L22 in archaea and eubacteria) exposes a long ␤-hairpin with a unique, twisted conformation (see Fig. 1D). Distal to the tip of the ␤-hairpin, the tunnel continues with more or less constant diameter to widen into the so-called vestibule ϳ20 Å before the tunnel exit (11). In archaea and eukaryotes, part of the vestibule wall is formed by Rpl39 (L39 in archaea). Rpl4 and Rpl17 possess surface exposed globular domains ( Fig. 1, A, C, and D). The globular domain of Rpl17 is arranged at the exit platform, and the globular domain of Rpl4 is positioned close to the subunit interface near the L1 protuberance. In yeast, Rpl4 and Rpl17 are essential proteins, whereas Rpl39 is dispensable. However, loss of Rpl39 causes severe growth defects similar to aminoglycoside sensitivity and cold sensitivity (12,13).
The ribosomal tunnel has important functions in the regulation of translation and in early protein biogenesis (9,14,15). On the one hand, the conformation of a nascent polypeptide is strongly affected by the tunnel environment. On the other hand, the nascent polypeptide conformation impacts the tunnel components, which in turn may transmit the information to distant sites of the ribosome. The exact mechanisms of the interplay between nascent chains, tunnel components, and other functionally important sites of the ribosome are not understood.
Some nascent polypeptides can adopt an ␣-helical structure within the ribosomal tunnel. This was determined with two different approaches that make use of the fact that polypeptide segments are more compact when they adopt an ␣-helical rather than an extended conformation. In the tunnel of wheat germ ribosomes, ␣-helix formation was monitored by incorporating two fluorescent dyes into the same nascent chain and monitoring separation of the dyes via FRET (16,17). In the tunnel of rabbit ribosomes, ␣-helix formation was tested via the availability of cysteine residues within the nascent chain for modifying reagents, which cannot enter the ribosomal tunnel (18 -20). In both systems, it was found that transmembrane (TM) segments can adopt an ␣-helical conformation within the eukaryotic ribosomal tunnel. A later cryo-EM study revealed that a hydrophilic nascent chain segment with a strong propensity to from an ␣-helix even free in solution indeed adopted ␣-helical conformation in the vestibule of the wheat germ tunnel (21).
Until now, studies of contacts between eukaryotic tunnel proteins and nascent chains are confined to the wheat germ system. In a previous study, it was shown that ribosomal proteins corresponding in size to Rpl4 (ϳ40 kDa), Rpl17 (ϳ20 kDa), and Rpl39 (ϳ6 kDa) contact a TM segment inside the tunnel (16). In a later study, the consequences of these interactions for translocation of multimembrane-spanning proteins into the endoplasmic reticulum membrane were characterized (17,22). In this context, Lin and co-workers (22) established the contact between TM segments and Rpl17 using an Rpl17-spe-cific antibody. Due to the lack of antibodies, similar experiments were not performed with respect to Rpl4 or Rpl39 (17). To date, contacts between the tunnel proteins and non-TM segments have not been analyzed in similar detail.
Here, we have employed a homologous yeast translation system, a newly developed FLAG-exposure assay, and site-specific UV cross-linking to identify and characterize interactions of nascent TM and non-TM segments with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 from yeast. As model nascent chains, we employed Dap2 and Dap2␣, which differ in a 16-residue segment only. Dap2 contains a hydrophobic SA, which forms an ␣-helical TM anchor, and Dap2␣ contains a hydrophilic segment with the propensity to form an ␣-helix. Using this set-up, we asked: does the SA segment fold in the ribosomal tunnel of yeast? Does folding occur in the vestibule or already in the narrower regions of the tunnel? Does a hydrophilic nascent chain segment with the propensity to form an ␣-helix also fold? How do the contacts of the TM and non-TM segments to the three tunnel proteins compare?

EXPERIMENTAL PROCEDURES
Strains and Plasmids-MH272-3f␣ (ura3, leu2, his3, trp1, ade2) (23) was used as a wild type strain. To generate a strain expressing exclusively His 6 -Rpl4a, we generated a ⌬rpl4a⌬rpl4b strain, in which both copies of RPL4 (RPL4A and RPL4B) were FIGURE 1. The ribosomal tunnel proteins of yeast. A, view of the Saccharomyces cerevisiae large ribosomal subunit based on PDB code 3U5H/3U5I (7,10). Shown is the ribosomal RNA (gray) of the large subunit and the three tunnel proteins Rpl4 (purple), Rpl17 (lime), and Rpl39 (gray). B, enlarged image section of the tunnel exit region. Left, exterior view; right, same image section but with a clipping plane at the constriction site of the tunnel. Rpl4 (purple, flexible residues in orange, insertion segment in yellow); Rpl17 (lime, flexible tip of the ␤-hairpin in green, hinges in brown). C, superposition of S. cerevisiae Rpl4 (colors are as described in A, PDB code 3U5I) and Haloarcula marismortui L4 (gray, PDB code 2QA4). The inset shows a close-up of the tunnel exposed segments of Rpl4/L4. The flexible segment of S. cerevisiae Rpl4 (orange) is structurally similar to the corresponding segment of H. marismortui L4. The insertion segment (yellow) leads to the formation of an extended loop in Rpl4. D, superposition of S. cerevisiae Rpl17 (color code as described in A, PDB code 3U5I) and H. marismortui L22 (gray, PDB code 2QA4) reveals structural similarity. E, partial amino acid sequences of S. cerevisiae Rpl4 (amino acids 59 -97) and S. cerevisiae Rpl17 (amino acids 116 -154), containing the tunnel exposed flexible segments. Rpl4, orange, flexible residues; yellow, insertion segment; Rpl17, green, flexible tip, brown, hinges.
deleted. To that end, RPL4A and RPL4B plus 300 base pairs upand downstream of the open reading frames were cloned into pSP65 (Promega). In the case of RPL4a, a HindIII/StuI fragment within the coding region was replaced with the LEU2 marker gene. In the case of RPL4b, a BstEII/MfeI fragment within the coding region was replaced with the HIS3 gene. The resulting disruption constructs were used to generate the diploid MH272-3f rpl4a::LEU2 rpl4b::HIS3 strain. Tetrad dissection revealed that haploid strains containing both mutations rpl4a::LEU2 and rpl4b::HIS3 were unviable (data not shown). The N-terminally His 6 -tagged version of RPL4a contains a 42-base pair insertion following the third codon of the RPL4a open reading frame encoding for the amino acids GSSHHHHHHSSGLV. In addition, codons four and five (numbering corresponding to wild type Rpl4a) were changed to GCTAGC to introduce a Nhe1 site. This results in the exchange of Q4L and V5A at the amino acid level. Expression of His 6 -Rpl4 from plasmid pYCPlac22 in a ⌬rpl4a⌬rpl4b background rescued the lethal phenotype of the double deletion strain (data not shown).
In Vitro Transcription and Translation Reactions-Yeast translation extracts were prepared as described previously (26) from MH272-3f␣ (wild type strain) or ⌬rpl4a⌬rpl4b ϩ pYC-Plac22-His 6 -Rpl4 (His 6 -Rpl4 strain). DNA templates for transcription reactions were generated by PCR using TaqDNA polymerase. The lengths of the nascent chains was determined by the reverse primers that generated truncated, stop codonless templates as described previously (25). mRNAs were generated by in vitro transcription using SP6 polymerase (26). Translations for UV cross-linking reactions were performed in the presence of [ 35 S]methionine (PerkinElmer Life Science) (24). Translations for the FLAG exposure experiments were performed in the presence of cold methionine. FLAG-tagged nascent chains were used to isolate ribosome nascent chain complexes (RNCs) under native conditions in the FLAG exposure assay, or to enrich UV cross-linking products prior to analysis under denaturing conditions.
FLAG Exposure Assay-FLAG-tagged RNCs were generated and isolated as described (25,26). In brief, 80-l translation reactions were incubated at 20°C for 80 min and were subsequently terminated by the addition of cycloheximide to a final concentration of 200 g/ml. In UV cross-linking experiments, samples were photolyzed prior to purification as described (24). Translation reactions were centrifuged at 95,000 rpm, at 4°C in a TLA100 rotor for 20 min. The pellet after centrifugation, containing ribosomes and RNCs, was carefully resuspended in 150 l of IP buffer (20 mM HEPES-KOH, pH 7.4, 120 mM potassium acetate, 2 mM magnesium acetate, 50 g/ml trypsin inhibitor, 1 mM PMSF, protease inhibitor mix (1.25 g/ml leupeptin, 0.75 g/ml antipain, 0.25 g/ml chymostatin, 0.25 g/ml elastinal, 5 g/ml pepstatin A). Forty l of anti-FLAG® M2 affinity gel (anti-FLAG beads, Sigma) were added, and the mixture was incubated for 1 h at 4°C on a shaker. Anti-FLAG beads were separated from the supernatant by centrifugation and were washed twice with 500 l of ice-cold IP buffer. Bound material was released by incubation in SDS-PAGE sample buffer for 10 min at 95°C, and subsequently, aliquots were run on 10% Tris-Tricine gels (27), transferred to nitrocellulose filters, and were analyzed by Western blotting using the antibodies indicated in the figure legends. Antibodies employed for decoration of Western blots and affinity purification of UV cross-links were raised in rabbits and described previously (25).
UV Cross-linking-For site-specific UV cross-linking experiments, the TAG amber mutation was introduced at specific positions of Dap2, FLAG-Dap2, and Dap2␣ and the photoactivatible cross-linker ⑀ANB-Lys-tRNA amb (tRNA Probes; termed "probe" hereafter) was incorporated at the TAG codon during in vitro translation as previously described (24). The position of the amber mutation indicates the distance of the probe from the N terminus of the nascent chain (see above). By changing the length of the nascent chain, probes were positioned at different distances from the PTC. As an example, 60-residue nascent Dap2 with the amber mutation at position 39 is termed Dap2-amb39-60; the distance of the probe from the PTC in this nascent chain is 21 residues (PTC-⌬21). In some experiments, UV cross-linking was performed with FLAG-tagged nascent chains to enrich nascent chain cross-links via FLAG affinity purification (FLAG-IP). Cross-linking reactions were not affected by the presence of the FLAG tag (data not shown). In UV cross-linking experiments, the residues of the FLAG tag were omitted in the calculation of the length of the nascent chain.
A standard 80 l of UV cross-linking reaction contained 48 pmol of ⑀ANB-Lys-tRNA amb . Translations were performed in the dark for 80 min at 20°C. Samples were then photolyzed in an ice-water bath for 10 min using a 500-watt mercury arc lamp (24). To identify cross-linking partners, samples were denatured, and subsequently, immunoprecipitations were performed under denaturing conditions using protein A-Sepharose beads coated with anti-Rpl17, anti-Rpl39, or anti-Srp54 as indicated (24). His 6 -Rpl4a extract was employed for the identification of the cross-link between the nascent chain and Rpl4. To this end, translations were performed in a translation extract derived from ⌬rpl4a⌬rpl4b expressing His 6 -Rpl4a (His 6 -Rpl4 extract). After UV cross-linking, translation reactions were denatured in dissociation buffer (200 mM Tris-HCl, 4% SDS, 100 g/ml bovine serum albumin, protease inhibitor mix (as described above), 1 mM PMSF, pH 7.5). Twenty-five l of the denatured samples were added to 30 l of Ni-NTA (Qiagen) in a volume of 600 l buffer N1 (20 mM Tris-HCl, 1% Triton X-100, 300 mM NaCl, pH 8.0). Samples were incubated for 2 h at 4°C, washed once with 600 l of buffer N1, once with 600 l of buffer N2 (20 mM Tris-HCl, 1% Triton X-100, 300 mM NaCl, 50 mM imidazole, pH 8.0), and finally were washed twice with 50 l of buffer N3 (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). Nascent chains and UV-adducts bound to Ni-NTA were released by incubation in SDS sample buffer for 10 min at 95°C. Samples were analyzed on 10% Tris-Tricine gels followed by autoradiography. In some experiments RNCs carrying radiolabeled FLAG-tagged nascent chains were isolated via anti-FLAG beads. In this case, the IP buffer was supplemented with 0.1% v/v Triton X-100. Anti-FLAG beads were washed once with 500 l of ice-cold IP buffer supplemented with 0.1% Triton X-100 and twice with 50 l ice-cold IP buffer lacking Triton X-100.

RESULTS
To test for ␣-helix formation within nascent chain segments inside the ribosomal tunnel, we developed the FLAG exposure assay. To that end, a sequence encoding for the eight-residue FLAG tag was inserted 3Ј of the start codon of the gene of interest ( Fig. 2A). Then, mRNAs of different lengths, each lacking a stop codon, were transcribed in vitro and were subsequently employed to produce RNCs in a yeast translation extract (25,28). Because affinity purification of RNCs under native conditions works only if the FLAG antibody can access the nascent FLAG tag, the approach allows determination of the effective length of the yeast ribosomal tunnel. Nascent chains of the soluble, cytosolic enzyme Pgk1 from yeast (29) were employed to determine the length of an extended nascent chain protected by the yeast ribosomal tunnel. To that end, [ 35 S]methionine-labeled RNCs carrying different lengths of FLAG-Pgk1 (Fig. 2B) were generated and similar quantities of RNCs were applied to FLAG-IP reactions (Fig. 2C, input RNCs). Western blotting with a ribosomal marker protein was then employed to determine whether RNCs were able to bind to the anti-FLAG beads (Fig. 2C, ribos via FLAG-IP). Using this FLAG exposure assay, RNCs carrying 46-as well as 50-residue FLAG-Pgk1 were efficiently isolated, however, RNCs carrying 40-residue FLAG-Pgk1 were not (Fig. 2C). Thus, the FLAG epitope was not sufficiently exposed on 40-residue nascent FLAG-Pgk1 but was exposed at a length of 46 amino acids or more.
Dap2 is a type II membrane protein, which contains a short, but strongly hydrophobic, SA localized between amino acids 30 -45 ( Fig. 2A). During the biogenesis of Dap2, the SA serves as the signal recognition particle (SRP)-dependent targeting signal and later serves as the single TM segment of Dap2 (24, 30 -33). To test for compaction of the SA with the FLAG expo- sure assay, the N-terminal 23 residues of Dap2 were replaced with a FLAG tag (FLAG-Dap2⌬N) (Fig. 2, A and B). As a result, 50 residues, but not 46 residues of FLAG-Dap2⌬N, were sufficient for the binding to the anti-FLAG beads (Fig. 2C). Mapping of residues one by one revealed that the minimal length of nascent FLAG-Dap2⌬N required for efficient exposure of the FLAG tag was 50 residues (Fig. 2D). Thus, FLAG-Dap2⌬N was more compact inside the tunnel than FLAG-Pgk1, most likely because the SA of Dap2 adopted an ␣-helical structure. The results confirm previous findings obtained with different experimental systems (16 -19, 21).
We next wanted to test whether secondary structure formation inside the tunnel also occurred in a hydrophilic segment, which can form a stable ␣-helix in a folded protein structure. To that end, we used the chimera Dap2␣, in which the 16-residue SA sequence was replaced with the complete 16-residue helix 5 of Pgk1 (Fig. 2, A and B) (24,29). The N-terminal 23 residues of Dap2␣ were replaced with a FLAG tag, such that FLAG-Dap2␣⌬N differed from FLAG-Dap2⌬N in only 16 residues (Fig. 2, A and B). Ribosome-bound FLAG-Dap2␣⌬N was efficiently isolated at a length of 45 residues (Fig. 2E). Thus, replacing the SA of Dap2 with helix 5 of Pgk1 changed the length of the nascent chain required for the exposure of the FLAG tag by five amino acids. Remarkably, the nascent chain length required for FLAG exposure showed a sharp cut-off for both FLAG-Dap2⌬N or FLAG-Dap2␣⌬N (Fig. 2, D and E). This indicates that the bulk of nascent chains of one kind had adopted the same, or very similar, conformation within the tunnel. Although the SA of FLAG-Dap2⌬N was compact, helix 5 of FLAG-Dap2␣⌬N was extended (Fig. 2, D and E).
To characterize the interactions of nascent chains with tunnel proteins in more detail, we wanted to unequivocally establish interactions of nascent chains with Rpl4, Rpl17, and Rpl39 in the yeast system. To that end, a UV-activatable probe was introduced at defined positions of nascent chains. This allowed us to test for the proximity of specific nascent chain residues with tunnel components (24).
First, the probe was introduced at position 39 of Dap2, and 60-residue radiolabeled nascent chains were generated (Dap2-amb39-60, Fig. 3A). The probe in Dap2-amb39-60 is localized 21 residues distal of the PTC (PTC-⌬21). After UV illumination, two specific cross-link bands were detected via autoradiography (Fig. 3B, red asterisks). The molecular mass of the upper band was consistent with a cross-link of Dap2-amb39-60 to Rpl4 that of the lower band was consistent with a cross-link to Rpl17. Indeed, the faster migrating cross-link was immunoprecipitated with anti-Rpl17 under denaturing conditions (Fig.  3C). Because the available Rpl4 sera did not efficiently immunoprecipitate Rpl4 under denaturing conditions (data not shown) a yeast strain was generated, in which both copies of Wild type RNCs carrying radiolabeled FLAG-tagged nascent chains, were photolyzed (ϩ UV), or, as a control, were kept in the dark (ϪUV). Subsequently, cross-linked products were purified via anti-FLAG beads and were analyzed on Tris-Tricine gels followed by autoradiography. The two major cross-linking products are labeled with a red asterisks. Positions of prestained molecular mass marker proteins are indicated. Note that the N-terminal FLAG tag on the nascent chain, which was employed to enrich the cross-link products, was not included in the calculation of the nascent chain length. C, identification of a cross-link between Rpl17 and Dap2-amb39-60. Wild type RNCs carrying radiolabeled Dap2-amb39-60 were photolyzed as described in B. Subsequently, cross-link products to Rpl17 were immunoprecipitated under denaturing conditions using anti-Rpl17 coupled to protein A-Sepharose beads (Rpl17-IP). xL17 indicates the cross-link product between Dap2-amb39-60 and Rpl17. D, identification of a cross-link between Rpl4 and Dap2-amb39-60. RNCs carrying radiolabeled Dap2-amb39-60 were generated in a wild type (wild type) or His 6 -Rpl4 (His 6 -Rpl4) translation extract. As a control, unmodified Lys instead of ⑀ANB-Lys was incorporated into the nascent chains (Ϫprobe). After photolysis, samples were subjected to Ni-NTA affinity purification. xL4 indicates the cross-link product between Dap2-amb39-60 and His 6 -Rpl4. The red asterisk indicates the cross-link to wild type Rpl4, which runs at a lower molecular mass. E, identification of cross-link between Rpl39 and Dap2-amb38-75. RNCs carrying radiolabeled Dap2-amb38-75 were generated in a wild type translation extract. After photolysis, cross-links to Rpl39 were immunoprecipitated using anti-Rpl39 coupled to protein A-Sepharose beads (Rpl39-IP). xL39 indicates the cross-link product between Dap2-amb39-60 and Rpl39. Analysis of the samples in C-E was as described in B. The total (tot) represents 10% of material employed for immunoprecipitation reactions (C and E) or Ni-NTA purification reactions (D). NOVEMBER 22, 2013 • VOLUME 288 • NUMBER 47

Interaction of Nascent Chains with Ribosomal Tunnel Proteins
Rpl4 (Rpl4a and Rpl4b) were replaced with hexahistidinetagged Rpl4a (⌬rpl4a⌬rpl4b ϩ His 6 -Rpl4). RNCs carrying Dap2-amb39-60 were then generated in a wild type or in a translation extract derived from the strain expressing His 6 -Rpl4 (His 6 -Rpl4 extract). The molecular mass of the cross-link obtained with His 6 -Rpl4 extract was increased compared with wild type (Fig. 3D). Moreover, the cross-link to His 6 -Rpl4, but not to Rpl4, was isolated via Ni-NTA affinity purification (Fig.  3D). Thus, a probe in position PTC-⌬21 within the SA was in contact either with Rpl17 or Rpl4. No cross-link to Rpl39 was detected. To test for the interaction of the SA with Rpl39 in the vestibule of the tunnel, we employed Dap2-amb38-75, in which the probe is positioned at PTC-⌬37 (Fig. 3A). In this nascent chain, the probe was in close contact to a protein of ϳ6 kDa, which was identified as Rpl39 via immunoprecipitation using anti-Rpl39 (Fig. 3E). Thus, all three tunnel proteins of yeast make contact to a TM segment, such as the SA of Dap2.
If a nascent chain segment inside of the tunnel adopts ␣-helical conformation, different scenarios with respect to its con-tact with tunnel proteins can be envisaged. At one extreme, a stable helical segment might move without changing its orientation with respect to the tunnel wall. Alternatively, the adjacent extended nascent chain segments might allow a helix to rotate around its axis, such that its orientation with respect to the tunnel wall changes. To distinguish between these possibilities, we employed a series of constructs in which the probe was placed at five successive positions between residue 39 (amb39) and residue 35 (amb35) of Dap2. The length of the nascent chains was then shortened successively such that the probe was at PTC-⌬21 in all nascent chains (Fig. 4A). If the SA adopted a stable ␣-helical conformation (Fig. 2), the probes would stick out to different sides, covering approximately one turn of an ␣-helix (Fig. 4A). This set of probes was used to test for proximity to Rpl4 and Rpl17, respectively. The results show that all of the probes at PTC-⌬21 were in close proximity to Rpl4 (Fig.  4B). However, only two probes, oriented to the same side of the helix, were in close proximity to Rpl17 (Fig. 4, A and C, Dap2-amb39-60 and Dap2-amb35-56). Thus, Rpl4 was in contact FIGURE 4. Sequence context and orientation of a probe at position PTC-⌬21 affects cross-linking to Rpl17 but not to Rpl4. A, schematic representation of Dap2-amb39-60, Dap2-amb38 -59, Dap2-amb37-58, Dap2-amb36 -57, and Dap2-amb35-56. The color code is as described in the legend to Fig. 3. B, contacts between Rpl4 and a probe in the SA at position PTC-⌬21. RNCs carrying radiolabeled nascent chains as shown in A were generated in a His 6 -Rpl4 translation extract. For direct comparison, RNCs carrying Dap2␣-amb39-60 were analyzed in parallel. As a control, unmodified Lys instead of ⑀ANB-Lys was incorporated into the nascent chains (Ϫprobe). After UV cross-linking samples were denatured, cross-link products were purified via Ni-NTA and were analyzed on a Tris-Tricine gel followed by autoradiography. The lower panel shows the relative amount of RNCs applied to the cross-linking reactions (input). The cross-link between nascent chains and Rpl4 is indicated in red (xL4). C, contacts between Rpl17 and a probe in the SA at position PTC-⌬21. RNCs carrying radiolabeled nascent chains shown as in A were generated in a wild type translation extract. After UV cross-linking samples were denatured, cross-link products were immunoprecipitated via anti-Rpl17-coupled beads and were analyzed on a Tris-Tricine gel followed by autoradiography. The cross-link between nascent chains and Rpl17 is indicated in red (xL17). D, schematic representation of Dap2␣-amb39-60, Dap2␣-amb38 -59, Dap2␣-amb37-58, and Dap2␣-amb36 -57. The color code is as described in the legend to Fig. 3, and helix 5 is indicated in blue. E, contacts between Rpl4 and a probe in helix 5 at position PTC-⌬21. Nascent chains shown in D were analyzed as described in B. For direct comparison, RNCs carrying Dap2-amb39-60 were analyzed in parallel. As a control, one sample was kept in the dark (ϪUV). F, contacts between Rpl17 and a probe in helix 5 at position PTC-⌬21. RNCs as shown in D were generated in a wild type translation extract and were analyzed as described in C. The UV cross-link between Rpl17 and Dap2-amb39-60 (lane 1) served as a positive control.
with all sides of the ␣-helical SA, whereas Rpl17 was in contact with only one side of the same helix.
Dap2␣ was employed to test for interactions of Rpl4 and Rpl17 with extended, hydrophilic nascent chain segments. To that end, the probe was successively incorporated at position PTC-⌬21 between residues 39 -36 of Dap2␣ (Fig. 4D). All of these probe positions were in contact with Rpl4 (Fig. 4E). In contrast, none of the probe positions were in contact with Rpl17 (Fig. 4F). Next, we wanted to establish the region of the tunnel in which the nascent chain was in contact with the three tunnel proteins. In the case of Rpl4 and Rpl17, we employed Dap2-amb39 because Dap2-amb39-60 was in contact with both of the two tunnel proteins (Fig. 3, C and D, and Fig. 4, B and C). The length of Dap2-amb39 was increased from 45 to 80 in steps of five residues (Fig. 5A). Rpl4 was not close to the probe at PTC-⌬6 (Dap2-amb39-45) proximal of the tunnel constriction (Fig. 5, A and B). However, Rpl4 was close to the probe starting at position PTC-⌬11 up to position PTC-⌬41 at the very exit of the tunnel (Fig. 5, A and B). To confirm that Rpl4 was able to contact a probe emerging from the tunnel, we made use of the well characterized interaction between the SA of Dap2 and the Srp54 subunit of the SRP (24, 33). As expected, Srp54 was in contact with the SA in Dap2-amb39-80, but not in Dap2-39-75 (Fig. 5, A and C). In the same experiment, Rpl4 was in close proximity to both nascent chains (Fig. 5C). Thus, Rpl4 can indeed contact a probe accessible also for SRP. In contrast, Rpl17 was in contact with the probe only in a narrow and confined region of the tunnel. Contact of Rpl17 to a probe in the SA was detected between PTC-⌬19 and PTC-⌬23 (Fig. 5D), but was not detected for a probe at PTC-⌬16 or PTC-⌬26 (Fig. 5E).
To define the region of the tunnel in which nascent chains were in contact with Rpl39, we employed Dap2-amb38 (Fig. 6A, compare with Fig. 3E). The probe in 70-and 75-residue Dap2-amb38, but not in 65-or 80-residue Dap2-amb38, was in close proximity of Rpl39 (Fig. 6B). Thus, Rpl39 was in contact with SA residues between PTC-⌬32 to PTC-⌬37, in the vestibule region of the tunnel. Proximity of Rpl39 to the probe in the SA was orientation dependent (Fig. 6C). Only a probe at position 37 or 38 but not at position 36 or 39 within the SA was in contact with Rpl39 (Fig. 6D). However, in contrast to Rpl17, the interaction of Rpl39 was not confined to residues within the SA, but Rpl39 was also in close contact with helix 5 of Dap2␣ (Fig. 6,  E and F). FIGURE 5. Rpl17 contacts the SA in a confined region, whereas Rpl4 contacts the SA throughout the tunnel. A, schematic representation of nascent Dap2-amb39 between 45 to 80 residues. The color code is as described in the legend to Fig. 3. B, Rpl4 is close to a probe between positions PTC-⌬11-PTC-⌬41. RNCs carrying FLAG-tagged radiolabeled nascent chains of the indicated length were generated in wild type translation extract. UV cross-linking and enrichment of the cross-link products was performed as described in the legend to Fig. 3. Shown is an autoradiography of the material isolated via FLAG-IP (upper panel) and of the RNCs employed for the cross-linking reactions (lower panel, input). The cross-link between the nascent chains and Rpl4 is indicated in red (xL4). C, a probe at position PTC-⌬41 at the exit of the ribosomal tunnel is in close contact to either Rpl4 or Srp54. RNCs carrying radiolabeled nascent Dap2-amb39 of 75 or 80 residues were generated in a His 6 -Rpl4 translation extract and UV cross-linking was performed as described in the legend to Fig. 3. As a control, unmodified Lys instead of ⑀ANB-Lys was incorporated into the nascent chain (Ϫprobe). Cross-link products were purified via Ni-NTA as described in the legend to Fig. 3. Cross-links between nascent chains and Srp54 were immunoprecipitated using ␣Rpl54 coupled to protein A-Sepharose beads (Srp54-IP). Samples were analyzed on a Tris-Tricine gel followed by autoradiography. Total (tot) represents 10% of the material employed in the immunoprecipitation reactions. The cross-link between the nascent chain and Rpl4 (xL4), the cross-link between the nascent chain and Srp54 (xLSrp54) are indicated in red. The lower panel shows the RNCs on a shorter exposure of the same gel. D, contacts between Rpl17 and probes between positions PTC-⌬19 -PTC-⌬23. RNCs carrying radiolabeled nascent Dap2-amb39 were generated in a wild type translation extract and were analyzed as described in the legend to Fig. 4. The cross-link between the nascent chain and Rpl17 (xL17) is indicated in red. The lower panel shows the relative amount of RNCs employed for the cross-linking reactions (input). E, contacts between Rpl17 and probes between positions PTC-⌬16 -PTC-⌬26. The experiment was performed and analyzed as described in the legend to Fig. 4, the cross-link between the nascent chain and Rpl17 (xL17) is indicated in red. The lower panel shows the RNCs on a shorter exposure of the same gel.

DISCUSSION
The minimal binding epitope of the FLAG M2 antibody consists of DYKD, whereas the four extraneous residues of the FLAG tag ( Fig. 2A) are thought to provide a spacer, improving accessibility and flexibility of the binding epitope (34). The calculations below are based on the assumption that the FLAG M2 antibody can bind efficiently to the DYKD epitope, when the full-length eight-amino acid FLAG tag (M-DYKDDDK) has emerged from the tunnel. Under this assumption, the yeast ribosomal tunnel covers 36 residues of a predominantly extended nascent chain. This is in good agreement with structural data of the eukaryotic ribosomal tunnel (9). A study using a bulky masstagging reagent revealed that 33 residues of an extended nascent chain were protected by the tunnel (19). Most likely, the difference of three residues is owed to the different experimental systems: the FLAG exposure assay may require a spacer longer than four residues (see above), the mass-tagging approach might detect nascent chains prior to their exit from the tunnel. Looking at it from a different angle, one may view the tunnel exit as an extended opening region.
We observed a sharp transition from non-binding to binding with the FLAG exposure assay. This observation is best explained by the assumption that the bulk of nascent chains of one kind had adopted the same or similar conformation and that this conformation was stable. Otherwise, one would expect that nascent chains too short to bind when folded but long enough to bind when unfolded, had been trapped by binding to the FLAG antibody, driving the equilibrium to the unfolded state.
There is debate about the tunnel region(s), in which secondary structure formation can occur. Using the mass-tagging approach, poly-alanine segments were found to compact very close to the PTC. This compaction was lost at the constriction but was regained in the vestibule (11). A later study revealed that ␣-helix formation close to the PTC was confined to artificial segments such as the poly-Ala, however, was not observed for example for a natural TM segment, which adopted ␣-helical structure only in the vestibule (20). A cryo-EM study visualized a hydrophilic nascent chain segment with strong propensity to form an ␣-helix in the vestibule but no other region of the  Fig. 3. B, contacts between Rpl39 and probes in the SA between PTC-⌬27-PTC-⌬42 as shown in A. RNCs carrying radiolabeled nascent Dap2-amb38 were generated in a wild type translation extract, and UV cross-linking and analysis was performed as described in the legend to Fig. 3. 10% of the material employed in the reaction (tot) and the immunoprecipitated (Rpl39-IP). The cross-link between the nascent chain and Rpl39 (xL39) is indicated. The cross-link between nascent chains and Rpl4 was cut off to improve the facility of inspection. C, schematic representation of Dap2-amb36-75, Dap2-amb37-75, Dap2-amb38-75, and Dap2-amb39-75. D, Rpl39 contacts only specific residues within the SA. Cross-linking, affinity purification, and analysis was performed with the RNCs shown in C as described in B. E, schematic representation of Dap2-amb39-75 and Dap2␣-amb39-75. F, Rpl39 contacts probes in the SA and also in helix 5. Cross-linking, affinity purification, and analysis was performed with RNCs as shown in E as described in the legend to Fig. 3. tunnel. Of note, 90-residue nascent Dap2 (DPAP-B) was not visible in the tunnel, suggesting that it had not adopted a defined conformation. However, in these experiments, the SA of Dap2 was far outside of the tunnel (21,35). In contrast, a FRET approach revealed that a TM segment already adopted compact structure close to the PTC prior to passage of the tunnel constriction (17). We find that compaction of the nascent chain occurred when the most C-terminal residue of the SA was at PTC-⌬14. At this distance, the SA just passed the constriction (36) and faced the ␤-hairpin of Rpl17. Thus, folding of the SA in the yeast ribosomal tunnel occurred significantly before the SA had reached the vestibule (Fig. 7A).
Considering that Rpl4 and Rpl17 expose domains to the very same region of the tunnel (Fig. 1B), they interact with nascent chains surprisingly differently. What allows the ␤-hairpin of Rpl17 to be strongly discriminative with respect to its contacts, whereas the patch-like domain of Rpl4 is not? The UV probe used to analyze contacts between nascent chains and tunnel proteins is highly reactive and forms cross-links with any adjacent amino acid. The probe was attached to a lysine, the side chain of which is flexible and can be anywhere in a half-sphere with the radius equal to a fully stretched side chain centered at its C ␣ . This radius is 7.8 A in the case of lysine, covalent linkage with the probe extends the radius to ϳ12 Å (37,38). Thus, probes at the exit may loop back into the vestibule. However, even then, the probe is far from the patch-like Rpl4 domain seen in the structures of the eukaryotic tunnel (Fig. 1, A and B, and Fig. 7) (7, 8). Also, the cross-linking pattern of Rpl17, which is thought to transduce nascent peptide-specific information to other parts of the ribosome, is peculiar (5,39). Most of the ␤-hairpin is rigid and attached to the tunnel wall. Only a short tip between two hinges is thought to possess high flexibility (Fig. 1, D and E) (5,39). These properties may explain why Rpl17 does not contact a probe in the lower part of the tunnel. However, due to the flexibility and reactivity of the probe, it is surprising that Rpl17 did not contact any of the probes in nascent Dap2␣ (Fig. 7B). How Rpl17 is excluded from nearby residues, whereas Rpl4 promiscuously contacts residues throughout the tunnel is not understood. However, the surprising difference between Rpl17 and Rpl4 is not confined to yeast. Johnson and co-workers (17) observed similar cross-linking patterns of TM segments to Rpl17 and a protein corresponding in size to Rpl4 in the wheat germ system. The authors suggested that each sample contained folded and unfolded TM segments and that the probe was therefore positioned at different tunnel locations (17). However, the FLAG exposure assay suggests that in the yeast system, nascent chains of one kind adopted a uniform and stable conformation (see above). Moreover, the lack of contacts between Rpl17 and a variety of positions within nascent Dap2␣ cannot be accounted for with multiple nascent chain conformations.
We speculate that the tunnel exposed domain of Rpl4 is flexible. Indeed, analysis of the static properties of four different bacterial tunnels revealed that the tunnel-exposed loop of bacterial L4 is flexible and may move freely in the lumen (39). The glycine-rich loop of yeast Rpl4 contains an insertion of 8 residues compared with its bacterial counterpart (Fig. 1, C and D). Possibly, the whole segment is more flexible in translating ribosomes than suggested by the crystal structures (Fig. 1, A-C, and  Fig. 7). The data suggest that structural changes in a translating  Fig. 1). Nascent chain residues within hydrophilic segments are shown in light gray, and residues within the hydrophobic signal anchor segment in dark gray or red. In the vestibule region of the tunnel, the nascent chain possesses flexibility. This is indicated by the gray arrows. A, contacts of a nascent chain containing a hydrophobic SA segment. Rpl4 contacts residues from PTC-⌬11 to PTC-⌬41 (⌬11-⌬41). This extended zone of Rpl4 contacts (purple shadow in the tunnel lumen) suggests that the tunnel exposed domain of Rpl4 is flexible. In addition, the nascent chain and the probe within the nascent chain may adopt different conformations. Rpl17 contacts amino acid residues within the folded SA segment in only a narrow region of the tunnel (⌬19-⌬23). Rpl39 contacts residues within the SA in the vestibule region (not shown in the model). A nascent chain containing an ␣-helical domain becomes accessible from the outside 41 residues apart from the PTC (⌬41). B, contacts of a hydrophilic nascent chain. Contacts of Rpl4 and Rpl39 to a hydrophilic nascent chain are similar to those of a nascent chain containing a hydrophobic SA (compare with A). In contrast, Rpl17 is excluded from contacts to a hydrophilic nascent chain (black barrier). This suggests nascent chain-induced conformational changes inside the tunnel, which change the accessibility of Rpl17. A mostly extended hydrophilic nascent chain becomes accessible from the outside 36 residues apart from the PTC (⌬36). The nascent chain is flexible in the vestibule (gray arrow) and e.g. a residue at PTC-⌬36 can contact either Rpl4 or Rpl39. For more details, see "Discussion." ribosome may also affect the ␤-hairpin of Rpl17. Indeed, specific regions of the tunnel seem to be more flexible than originally thought (39). Specifically the region at PTC-⌬18 -22, in which we observe contacts between the SA and Rpl17, was recently found to undergo nascent chain induced conformational changes (36) and nascent chain interactions with ribosomes also alter the conformation of tunnel-exposed ribosomal RNA nucleotides (40).
Johnson and co-workers (17) found that a protein, corresponding in size to Rpl39, formed UV cross-links with the same probe positions that also formed cross-links to Rpl17. In our analysis, none of the probes was in contact with both Rpl17 and Rpl39. Consistent with its localization in the vestibule, Rpl39 was in contact only with probes between PTC-⌬32 to -⌬37 (Fig.  7). In the vestibule, the folded SA possessed a defined orientation with respect to Rpl39, whereas its orientation was random with respect to Rpl4. This observation is consistent with the idea that domains of Rpl4 possessed high flexibility, whereas Rpl17 and Rpl39 occupied more defined positions inside the ribosomal tunnel.