Translation regulation via nascent polypeptide-mediated ribosome stalling

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ScienceDirect Translation regulation via nascent polypeptide-mediated ribosome stalling Daniel N Wilson1,2, Stefan Arenz1 and Roland Beckmann1,2 As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit. Interaction between the nascent polypeptide chain and the ribosomal tunnel can modulate the translation rate and induce translational stalling to regulate gene expression. In this article, we highlight recent structural insights into how the nascent polypeptide chain, either alone or in cooperation with cofactors, can interact with components of the ribosomal tunnel to regulate translation via inactivating the peptidyltransferase center of the ribosome and inducing ribosome stalling. Addresses 1 Gene Center and Department of Biochemistry, Feodor-Lynenstr. 25, 81377 Munich, Germany 2 Center for Integrated Protein Science, Munich (CiPSM), Feodor-Lynenstr. 25, 81377 Munich, Germany Corresponding authors: Wilson, Daniel N ([email protected]) and Beckmann, Roland ([email protected])

Current Opinion in Structural Biology 2016, 37:123–133 This review comes from a themed issue on Macromolecular Machines and Assemblies Edited by David Barford and Karl-Peter Hopfner

http://dx.doi.org/10.1016/j.sbi.2016.01.008 0959-440/# 2016 Elsevier Ltd. All rights reserved.

at 5.8–6.1 A˚ resolution, which revealed electron density for the TnaC and DP120 NCs from the PTC to the tunnel exit [5,6], as originally predicted by Lake and coworkers in the 1980s [7,8]. These studies also suggested that both the NCs adopted extended conformations with approximately 30 amino acids required to span the ribosomal tunnel, consistent with earlier studies showing that eukaryotic and bacterial ribosomes protect more than 30 amino acids of the NC from proteolysis [9,10,11]. Subsequent cryo-EM studies of ribosome-NC complexes (RNCs) have revealed more compacted or a-helical-like NC conformations within distinct regions of the ribosomal tunnel [12,13], consistent with prior biochemical and biophysical studies [14,15,16,17,18,19]. While the dimensions of the tunnel allow a-helix formation, the folding of domains as large as an IgG domain (17 kDa) seems less feasible [14,20]. Nevertheless, the formation of tertiary structure, such as a- and b-hairpins, has reported to occur near the tunnel exit (>80 A˚ from tunnel start) where the tunnel widens to form a vestibule [19,21,22]. Moreover, recently, the folding of small zinc finger domain consisting of 29 amino acids from the ADR1 protein was observed to fold within the ribosomal tunnel [23]. Specifically, the short a-helix and b-hairpin were observed to fold around a zinc ion within the last 60–80 A˚ of the tunnel, that is, just below the uL22/uL4 constriction, but before the tunnel vestibule [23].

The influence of the nascent polypeptide chain on translation The nascent polypeptide chain within the ribosomal tunnel The ribosome is the protein synthesizing machine of the cell, polymerizing the nascent polypeptide chain (NC) from its substituent amino acid building blocks. The active site for peptide bond formation is the peptidyltransferase center (PTC), which is located in a cleft on the intersubunit side of the large ribosomal subunit [1,2]. As the NC is being synthesized, it traverses a tunnel within the large subunit and exits at the solvent side where protein folding and targeting events occur [3]. Accumulating evidence has revealed that rather than being a passive conduit for the NC, the ribosomal tunnel plays a more active role in early protein folding events as well as in the regulation of translation [3,4]. The first direct visualization of the NC within the ribosomal tunnel came from cryo-electron microscopy (cryo-EM) reconstructions www.sciencedirect.com

Peptide bond formation at the PTC involves the accurate placement of the substrates to allow nucleophilic attack of the a-amino group of the aminoacyl-tRNA in the A site onto the carbonyl-carbon of the peptidyltRNA in the P site [1,2]. While the rate of this nucleophilic attack varies for each amino acid [24], specific amino acids or combinations of amino acids disfavour peptide bond formation to such an extent that they slow translation and can even promote translational arrest [4]. In eukaryotes, consecutive stretches of positively charged amino acids, such as lysine or arginine, have been shown to induce translational arrest [21,25,26,27,28]. In addition, proline is both a poor A-site acceptor of the peptidyl moiety [29,30] as well as poor donor when located in the P site [24,31,32]. Moreover, proline-containing motifs promote ribosome stalling during translation elongation and termination, leading to subsequent tmRNA-mediated tagging [33,34,35]. Similarly, ribosome profiling experiments Current Opinion in Structural Biology 2016, 37:123–133

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in mammalian cells identified a number of prolinecontaining tripeptide motifs as sites of ribosome accumulation [36]. Recent studies revealed that ribosome stalling is most prolonged when stretches of three or more consecutive proline residues occur in proteins [32,35,37,38]. Ribosome stalling at proline-containing motifs is influenced by the context of the NC, in particularly the amino acids directly flanking the proline residues [39,40,41,42,43]. Proline is also present in many leader peptide sequences that are known to induce programmed translational stalling and recent cryo-EM structures have provided the first high resolution insights into the mechanism by which the NC cooperates with components of the ribosome to silence the PTC and induce ribosome stalling.

Nascent polypeptide mediated translational stalling A number of NCs, or so-called NC arrest sequences (ASs), induce translational stalling to regulate expression of a downstream gene [4] (Figure 1). In contrast to proline-rich antimicrobial peptides that act in trans to inhibit translation by binding within the ribosomal tunnel [44,45], ASs act in cis on the ribosome during their own translation to induce ribosome stalling. Generally, the AS contains a window of approximately 20 amino acids that stall translation via direct interaction with components of the ribosomal tunnel. Depending on the AS, the translation arrest can occur either during translation elongation when a sense codon is present in the A-site, for example SecM, MifM, VemP, CatA86L,

Figure 1

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Transcription antitermination via translation termination stalling Rho Translation stalling

Rho-dependent transcription termination

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Regulation of gene expression by ribosomal stalling. (a) Translation termination stalling on the TnaC leader peptide causes the ribosome to block Rho binding sites and thus preventing Rho-dependent transcription termination. Transcription of the downstream tnaA/B genes allows their expression via internal translation initiation. (b) Stalling during translation elongation of SecM, MifM, VemP, ErmCL, and CatA86 leader peptides causes the ribosome to block stem-loop formation and exposes the ribosome binding site (RBS) of the downstream cistrons, allowing their expression. (c) Stalling during translation termination of upstream open reading frames (uORFs) of arginine attenuator peptide (AAP), cytomegalovirus (CMV) and S-adenosyl-methionine decarboxylase (SAM-DC) prevents scanning and therefore represses expression of the respective downstream genes. Current Opinion in Structural Biology 2016, 37:123–133

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Ribosome stalling Wilson, Arenz and Beckmann 125

ErmCL, or during translation termination when a stop codon is present in the A-site, for example TnaC, AAP, CMV and SAM-DC uORF (Figure 1) [4]. The translational arrest during elongation may occur at a defined site, as seen for ErmCL [46,47] or VemP [48], or at multiple sites as observed for MifM and SecM [49,50]. It should also be noted that some ASs induce translation arrest during both elongation and termination, such as ErmCL, MifM, and AAP, indicating that both the peptidyl-transferase activity during elongation and peptidyl-hydrolyse activity during termination are inhibited. In other cases, it has been shown that the translational arrest is specific for termination, for example TnaC and the uORF of SAM-DC, or that the strength of the translational arrest is dependent on the nature of the A-site amino acid, for example, ErmAL1 [51]. Given the conservation of the ribosome, it is not surprising that ASs from one species can induce ribosome stalling on the ribosomes of another species, for example, ribosome stalling during translation of Arabidopsis thaliana CGS1 or AAP from the fungi Neurospora crassa is also observed in vitro on rabbit reticulocyte and wheat germ ribosomes [52,53]. Similarly, gp48/UL4 uORF2 from the human cytomegalovirus (hCMV) not only induces ribosome stalling on human ribosomes [54], but also in rabbit, wheat germ, Drosophila and yeast translation systems [13]. In some cases, the ribosome stalling induced by the AS is species-specific, with the best characterized examples being MifM and SecM; MifM stalls in the Gram-positive bacteria Bacillus subtilis but not in the Gram-negative Escherichia coli, whereas the opposite effect is observed with SecM [55]. Generally, stalling during translation of bacterial uORFs leads to up-regulation of expression, either (i) via anti-termination, whereby translation arrest causes the ribosome to block the binding sites for the Rho transcription terminator, leading to transcription and thus translation of the downstream genes, for example TnaC [3,4] (Figure 1a), or (ii) by inducing a conformational rearrangement in the mRNA that exposes the Shine-Dalgarno sequence and thus allows ribosome binding and subsequent translation of the downstream genes, for example, SecM, MifM, VemP, ErmCL, Cat86L (Figure 1b). In contrast, stalling during translation of the eukaryotic uORFs leads to repression by preventing ribosome scanning and initiation at the downstream gene, for example, AAP, CMV, SAM-DC [3,4] (Figure 1c). The stalling capability of leader peptides is not always intrinsic to the AS, but can instead require an additional extrinsic co-effector molecule, for example, AAP [56] and TnaC [57] require the amino acids arginine and tryptophan, respectively, whereas Cat86L and ErmCL require the presence of the antibiotics chloramphenicol and erythromycin, respectively [58,59]. In addition, polyamines induce ribosome stalling during translation of the uORF of SAM-DC [3,4] (Figure 1c). www.sciencedirect.com

Recently four cryo-EM structures have provided structural insight into how NC arrest sequences in the ribosomal tunnel can induce ribosome stalling. Specifically, two cryo-EM structures illustrate how the NC can arrest translation in a co-factor independent manner, namely, the human ribosome stalled on the CMV gp48/UL4 uORF2 sequence [54] and the B. subtilis ribosome stalled on the MifM leader peptide [60]. Additionally, two cryo-EM structures illustrate how the NC can cooperate with a co-factor to arrest translation, namely, the E. coli ribosome stalled on either the TnaC leader peptide in the presence of tryptophan [61] or the ErmCL leader peptide in the presence of erythromycin [62].

Co-factor independent stalling on the CMV UL4 uORF2 An example of a co-factor independent NC arrest sequence is found in the upstream uORF2 of the gp48/UL4 gene of the hCMV [63,64]. In this case, ribosome stalling occurs during translation termination of uORF2 [65,66], which prevents ribosomes from scanning and initiating at the downstream gp48/UL4 gene [63,64] (Figure 1c). There is no release mechanism known for this staller, which therefore leads to a general down regulation of the gp48/UL4 gene expression under normal conditions. Yet, when canonical translation initiation is globally down regulated leaky scanning would allow for bypassing of the uORF2, thus, resulting in an enhanced and selective expression of the downstream gp48/UL4 gene. uORF2 encodes 22 amino acids of which the C-terminal Pro21 and Pro22, located directly before the UAA stop codon, are absolutely critical for the translation arrest [54,64,67]. Other critical residues are Ser12 and Ala8Ser7 that delineate the stretch of contributing amino acids towards the N-terminal end of the peptide [64,67]. The cryo-EM structure of a uORF2-stalled human ribosome revealed the structural details of the peptide in the tunnel and its influence on the PTC [54] (Figure 2a). Most unexpectedly, the peptide forms an a-helix in the upper part of the tunnel before the two critical C-terminal prolines (Figure 2b). The helix establishes several contacts to residues of the tunnel wall resulting in a central positioning. The helix ends at the tunnel constriction formed by the ribosomal proteins uL4 and uL22, with the most distal His133 of the uL22 b-hairpin in close vicinity to the last critical residues Ser7-Ala8 of the uORF2 (Figure 2b). As a consequence of this helix positioning the C-terminal prolines are stabilized in a distinct conformation that deviates from the canonical path of nascent chains observed so far. This in turn results in a reorientation of the otherwise clashing base of residue U2585 (E. coli numbering) (Figure 2c). U2585 (U4494 in H. sapiens) rotates by 908 away from the PTC (Figure 2c), and is thereby unable to participate in any reactions at the PTC. This distinct conformation of U2585 is likely to provide an explanation for the lack of termination activity at the CMV termination codon since mutations of this base lead Current Opinion in Structural Biology 2016, 37:123–133

126 Macromolecular Machines and Assemblies

Figure 2

(a) CMV

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Cryo-EM structures of CMV- and MifM-stalled ribosomes. (a) Overview of CMV-RNC with small subunit (yellow), large subunit (grey), eRF1 (pink), P-site tRNA (green) and the CMV AS (blue) [54]. (b) Contacts of the nascent chain (blue) with 28S rRNA nucleotides (E. coli numbering) of the ribosomal tunnel and with r-protein L22 (cyan). (c) Comparison of positions of U2585 in the CMV-RNC with the respective position in the human post-state ribosome [91]. (d) Overview of MifM-RNC with small subunit (yellow), large subunit (grey), P-site tRNA (green) and the MifM AS (orange) [60]. (e) Contacts of MifM (orange) with 23S rRNA nucleotides (E. coli numbering) of the ribosomal tunnel and with r-proteins uL22 (cyan) and uL4 (teal). (f) Comparison of 23S rRNA nucleotides U2506, U2585 and A2602 in MifM-RNC with their respective positions in the ribosomal prepeptide bond formation state [88]. The position A-site tRNA CCA-end is superimposed in transparent grey.

to loss of termination activity [68]. Perturbation of the PTC at this site as a cause for termination stalling is in agreement with the observation that the overall conformation of both the peptidyl-tRNA in the P-site as well as of the release factor (eRF1) with respect to the conformation of the critical GGQ motif appears to be regular [54]. Indeed, replacement of the stalling stop codon by a sense codon for alanine relieves stalling and allows continuation of translation elongation [54], consistent with the lesser importance of U2585 for peptidyltransferase activity. However, truncated CMV is also puromycin resistant indicating that translation elongation is also affected to some extend due to the perturbed PTC [69]. Taken together, in the case of the uORF2 of hCMV the nascent chain adopts a distinct position in the ribosomal tunnel that in combination with the geometrically Current Opinion in Structural Biology 2016, 37:123–133

challenged C-terminal proline pair results in a specific PTC perturbation and thus inactivation.

Species-specific translation arrest (MifM) In B. subtilis, the MifM leader peptide is located upstream of the gene encoding YidC2 (Figure 1b), a homolog of the constitutively expressed SpoIIIJ (YidC1), which is involved in membrane protein insertion and folding [70]. MifM contains a C-terminal region (residues 69–89) that is critical for ribosome stalling and an N-terminal transmembrane (TM) segment that targets the MifM NC for membrane insertion, presumably via SpoIIIJ [71]. Interaction between SpoIIIJ and the TM segment of MifM as it emerges from the ribosomal tunnel is thought to prevent ribosome stalling by providing a pulling force on the MifM nascent chain [55], analogous to SecA relief of www.sciencedirect.com

Ribosome stalling Wilson, Arenz and Beckmann 127

SecM stalling [72,73,74]. Subsequently, canonical translation termination and ribosome recycling ensues, leading to rapid refolding of the mRNA and repression of YidC2 expression. In contrast, when cellular levels of SpoIIIJ are low, ribosome stalling occurs on the MifM leader peptide, maintaining the unfolded conformation of the mRNA and thereby promoting expression of YidC2. In this manner, B. subtilis ensures that sufficient levels of SpoIIIJ or YidC2 are present in the membrane to direct membrane protein insertion and/or protein folding [71,75]. Biochemical studies have demonstrated that four major ribosomestalling sites are present in the MifM AS; the first occurring when the codon for residue D89 is present in the Psite, then ribosomes stall at the following three codon positions corresponding to residues A90, G91 and S92 [49]. Mutagenesis studies have identified six residues (R69, I70, W73, I74, M80 and N81) as well as the negatively charged DEED sequence (residues 86–89) within the C-terminal region of MifM that are important for ribosome stalling [49,71]. Despite the high conservation of the ribosomal tunnel, translational stalling by MifM occurs on B. subtilis ribosomes, but not efficiently on E. coli ribosomes [55]. A cryo-EM reconstruction of a MifM-stalled B. subtilis ribosome has provided structural insight into the basis for MifM-mediated translation arrest as well as the species-specificity of the ribosome stalling [60] (Figure 2d). The MifM nascent chain was shown to adopt a predominantly extended conformation and to make extensive interactions with the ribosomal tunnel at the constriction area between uL4 and uL22 (Figure 2e). Of all the contacts observed between MifM and components of the ribosomal tunnel, only the b-hairpin of ribosomal protein uL22 is less conserved and exhibits species-specific differences. Subsequent biochemical analysis revealed that replacing the b-hairpin of B. subtilis uL22 with the equivalent E. coli sequence abrogated MifM stalling, but that a single reversion of Lys90 back to Met90 (as in B. subtilis uL22) restored MifM stalling activity [60], thus suggesting that residue 90 of uL22 can modulate the speciesspecificity of MifM stalling. In addition, the cryo-EM structure revealed that the conformation of the side chain of Glu87 of MifM, part of the ‘DEED’ motif, prevents PTC nucleotides U2506, and thereby also U2585, from adopting the conformations required for A-tRNA accommodation (Figure 2f). In the absence of any obvious relay of rRNA conformational changes from tunnel nucleotides to the PTC, it seems plausible that the interactions between MifM and the tunnel components, such as uL22, promote a defined conformation of MifM NC that orients the sidechain of residue Glu87 of MifM to interact and stabilize U2506 in an inactive state, which in turn prevents peptide bond formation by perturbing accommodation of the incoming aminoacyl-tRNA at the A-site. www.sciencedirect.com

Co-factor mediated translational arrest (TnaC) The TnaC peptide is a well-documented example of a bacterial AS that stalls the ribosome dependent on the presence of a small molecule, namely the amino acid Ltryptophan (L-Trp), in order to up-regulate specific genes [57]. Elevated levels of free L-Trp call for the expression of the enzymes tryptophanase (TnaA) and tryptophan-specific permease (TnaB) that are encoded together with the TnaC peptide in the tna operon (Figure 1a). A spacer between tnaC and tnaA contains Rho binding sites that are no longer accessible upon LTrp dependent translational stalling, thus, resulting in anti-termination of transcription and induction of tnaA and tnaB in the presence of elevated levels of free L-Trp [76] (Figure 1a). Interestingly, the TnaC peptide stalls naturally during translation termination, and mutation of the stop codon to a sense codon abrogates stalling [76]. TnaC uses a C-terminal proline which is critical for stalling [57], similar to the case of the uORF2 of hCMV. Several additional amino acids and their relative position to each other have been characterized to be important in this peptide [57,77,78]. Since several mutations in the ribosomal tunnel have also been shown to modulate stalling [57,77,78], it has been suggested that the nascent TnaC peptide and the ribosomal tunnel cooperate in monitoring free L-Trp levels. The cryo-EM analysis of a TnaC-stalled ribosome [61] indeed confirmed this (Figure 3a): First, the critical residues of the nascent peptide were found in a defined conformation in the ribosomal tunnel establishing numerous contacts to the tunnel wall reaching from the PTC all the way down to the uL22/uL4 constriction. Second, together with the peptide, two L-Trp molecules were present in the tunnel, apparently coordinated in composite binding pockets formed by ribosomal tunnel residues and the TnaC peptide (Figure 3b): While the first L-Trp molecule is buttressed between U2586 of the ribosome and residues 19–21 of the peptide, the second L-Trp molecule binds within a pocket formed by the peptide and ribosomal residues A2058 and A2059—also representing the interaction site for macrolides that block the ribosomal tunnel. The PTC silencing mechanism is therefore of an allosteric nature and employs a subtle relay system to modulate the PTC in a manner dependent on the TnaC peptide and small molecule interactions in the tunnel. The PTC, in particular 23S rRNA nucleotides A2602 and U2585, were found stabilized in a conformation that would not allow for productive release factor accommodation (RF2 in this case) (Figure 3c) [61]. Taken together, the TnaC peptide closely cooperates with the ribosome in order to create composite binding sites in the tunnel specific for L-Trp. Stabilization of the peptide in the presence of the small molecules is communicated allosterically to the PTC where a C-terminal proline together with the induction of an unfavorable geometry of the PTC for release factor accommodation as well as peptide transfer results in stalling. Current Opinion in Structural Biology 2016, 37:123–133

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Figure 3

(a) TnaC

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Current Opinion in Structural Biology

Cryo-EM structures of TnaC- and ErmCL-stalled ribosomes. (a) Overview of TnaC-RNC with small subunit (yellow), large subunit (grey), P-site tRNA (green) and the TnaC AS (pink) [61]. (b) Contacts of TnaC (pink) with 28S rRNA nucleotides (E. coli numbering) of the ribosomal tunnel including two molecules of free L-tryptophan (orange). (c) Comparison of positions of A2602 in the TnaC-RNC with the respective position in the RF2-bound ribosome [92]. (d) Overview of ErmCL-RNC with small subunit (yellow), large subunit (grey), P-site tRNA (green) and the ErmCL AS (green) [62]. (e) Contacts of ErmCL (green) with 23S rRNA nucleotides of the ribosomal tunnel. (f) Comparison of 23S rRNA nucleotide U2585 in ErmCL-RNC with its respective position in the ribosomal pre-peptide bond formation state [88]. The position A-site tRNA CCA-end is superimposed in transparent grey.

Drug-dependent translational stalling (ErmCL) Drug-dependent translational stalling is utilized by a variety of bacteria to regulate expression of antibiotic resistance genes in response to the accumulation of the drug in the cell [59]. For example, macrolide-dependent stalling during translation of Erm-type leader peptides induces expression of the Erm-type methyltransferases, which mono- or di-methylate A2058 of the 23S rRNA and confer resistance to the macrolide class of antibiotics by reducing their affinity for the ribosome [46]. Macrolide antibiotics bind within the ribosomal exit tunnel and inhibit translation of most proteins by blocking the path of the elongating NC. Recent studies, however, have revealed that NCs manage to bypass the drug in the tunnel and can even become fully synthesized in the presence of the drug [79,80,81]. Methylation of A2058 has been shown to incur Current Opinion in Structural Biology 2016, 37:123–133

a fitness cost to bacteria, presumably due to deregulation of translation [82], which is minimized by inducing expression of the Erm-type methyltransferases only when the drug is present [59]. One well-characterized example is the ErmCL arrest sequence, which induces stalling in the presence of the macrolide erythromycin and thereby induces expression of the erythromycin resistance methyltransferase ErmC (Figure 1b): Biochemical analyses have demonstrated that in the presence of erythromycin polymerization of ErmCL stalls because the ribosome is unable to catalyze peptide bond formation between the nine amino acid long ErmCL-tRNAIle (codon 9) in the ribosomal P-site and Ser-tRNASer (codon 10) in the A-site, and that ribosome stalling results from an intimate interplay between ErmCL, erythromycin and components of the ribosomal tunnel [47]. www.sciencedirect.com

Ribosome stalling Wilson, Arenz and Beckmann 129

Figure 4

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Common and distinct features of ribosome arrest sequences. (a) Schematic illustrating the common features of ribosome arrest sequences, which include (1) conformational rearrangements of PTC nucleotides, (2) proline residues within the NC at the PTC, (3) interactions of the AS with ribosomal components at the constriction site, and (4) relays either through the ribosome and/or NC to silence the PTC. (b)–(e) Schematic illustrating the distinct features of ribosome arrest sequences of (b) MifM, (c) CMV, (d) TnaC and (e) ErmCL.

This interplay has recently been visualized using cryoEM [62] revealing that the ErmCL nascent chain directly senses the tunnel-bound drug and thereby adopts a defined state that induces allosteric conformational rearrangements within the petidyltransferase center of the ribosome (Figure 3d). Multiple contacts are observed between the conserved C-terminal ‘IFVI’ (I6–I9) motif of ErmCL and components of the ribosomal tunnel, specifically, U2506 interacts with Val8 and stacks upon the aromatic side chain of Phe7, whereas Ile6 interacts with U2586. Consistently, alanine mutations of the ‘IFVI’ motif severely reduce ribosome stalling [47,83]. Direct interaction is also observed between Phe7 of ErmCL and the cladinose sugar of erythromycin (Figure 3e). Biochemical experiments support ErmCL monitoring for the presence and the structure of the drug. For example, ErmCL-stalling was observed in the presence of other cladinose-containing macrolides, but not in the presence of ketolide antibiotics, such as telithromycin, which lack the C3-cladinose [47,84]. Moreover, macrolides bearing www.sciencedirect.com

modifications of the C3-cladinose are also impaired for ErmCL-mediated ribosome stalling [84]. Surprisingly, U2585 adopts an unusual conformation in the ErmCL-stalled RNC, such that it is rotated by 808 compared to the canonical positions observed during peptide bond formation (Figure 3f). The rotation places U2585 in a pocket formed by U2584/G2583 and G2608, reminiscent of the conformation of U2585 observed in the CMV-stalled ribosome [54], as well as upon streptogramin binding to the Escherichia coli ribosome [85]. The canonical positions of U2585 sterically clash with the path of ErmCL, suggesting the conformation of the ErmCL NC itself may be the reason for the flipped state of U2585. During binding and accommodation of the A-tRNA, the ribose 20 -OH of A76 maintains hydrogen bonding distance to the C4 oxygen of U2585 [86,87,88], suggesting that the rotated conformation of U2585 contributes to preventing stable binding and accommodation of the aminoacyl-tRNA at the A-site, thus leading to inhibition of peptide bond formation and translation arrest [62]. Current Opinion in Structural Biology 2016, 37:123–133

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Conclusion

References and recommended reading

Taken together, the recent high-resolution structures of AS-stalled ribosomes provide a first glimpse at the common and specific features of their underlying molecular mechanisms.

Papers of particular interest, published within the period of review,

First, several aspects have been identified that all or most stallers have in common (Figure 4a): (i) In all cases the geometry of the PTC is perturbed in order to achieve silencing. Here, three nucleotides of the PTC, A2602, U2585 and U2506, are particularly prone to be affected due to their critical contribution to peptide bond formation and/or termination. Additionally, as part of the PTC perturbation, the CCA-ends of the A- and/or P-tRNAs can be repositioned which can also contribute to an inactive PTC geometry. (ii) Prolines are critical residues in many, but not all ASs, presumably due to their already poor reaction kinetics and restricted geometry [30,31,32,89]. (iii) All stallers establish specific contacts to the wall of the ribosomal tunnel, however, the critical regions appear to be concentrated on the upper half of the tunnel extending from the PTC to the uL22/uL4 constriction. (iv) Allosteric relay systems are proposed to transmit the molecular recognition events from the tunnel to the PTC, either through the nascent chain or via rRNA nucleotides comprising the tunnel. Second, apart from the above-mentioned aspects there appears to be no simple consensus for stalling, but rather a high degree of versatility (Figure 4b–e): (i) Different ASs stall elongation and/or termination with the critical amino acid stretch ranging from as short as three amino acids [90] to as long as 99 amino acids [48]. (ii) Stalling during elongation can occur at a distinct single site (TnaC, ErmCL) or over an entire stretch of amino acids (MifM, SecM). (iii) There is no consensus and no common contact pattern between the nascent chain and the ribosomal tunnel wall, with some ASs even adopting secondary structure in tunnel (Figure 4c). (iv) Small molecule sensing ASs participate in composite binding pockets together with the ribosomal tunnel (Figure 4d) while force sensing ASs impart a perturbed geometry at the PTC that is corrected and released due to application of force. Finally, some ASs lead to reversible stalling while others are dead-end stallers that become substrates for decay pathways. To obtain a more comprehensive picture of the commonality and diversity of mechanisms involved in nascent polypeptide-mediated translational stalling, further structural insight into other AS-stalled ribosomes will be required.

Acknowledgements Research in the Beckmann and Wilson labs is supported by the Deutsche Forschungsgemeinschaft SFB646 (to R.B.) and WI3285/4-1 (to D.N.W.) and GRK1271/FOR1805 (D.W and R.B.), as well as the Graduate School of Quantitative Biosciences Munich and the European Research Council (to R.B.). Current Opinion in Structural Biology 2016, 37:123–133

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