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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

(a)

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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50S Current Opinion in Structural Biology

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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(b)

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ErmCL Current Opinion in Structural Biology

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

have been highlighted as:  of special interest  of outstanding interest 1.

Simonovic M, Steitz TA: A structural view on the mechanism of the ribosome-catalyzed peptide bond formation. Biochim Biophys Acta 2009, 1789:612-623.

2.

Rodnina MV: The ribosome as a versatile catalyst: reactions at the peptidyl transferase center. Curr Opin Struct Biol 2013, 23:595-602.

3.

Wilson DN, Beckmann R: The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling. Curr Opin Struct Biol 2011, 21:1-10.

4. Ito K, Chiba S: Arrest peptides: cis-acting modulators of  translation. Annu Rev Biochem 2013, 82:171-202. Comprehensive review on nascent polypeptide mediated translation regulation. 5.

Becker T, Bhushan S, Jarasch A, Armache JP, Funes S, Jossinet F, Gumbart J, Mielke T, Berninghausen O, Schulten K et al.: Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 2009, 326:1369-1373.

6.

Seidelt B, Innis CA, Wilson DN, Gartmann M, Armache JP, Villa E, Trabuco LG, Becker T, Mielke T, Schulten K et al.: Structural insight into nascent polypeptide chain-mediated translational stalling. Science 2009, 326:1412-1415.

7.

Bernabeu C, Lake JA: Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain. Proc Natl Acad Sci U S A 1982, 79:3111-3115.

8.

Bernabeu C, Tobin EM, Fowler A, Zabin I, Lake JA: Nascent polypeptide chains exit the ribosome in the same relative position in both eukaryotes and prokaryotes. J Cell Biol 1983, 96:1471-1474.

9.

Malkin LI, Rich A: Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J Mol Biol 1967, 26:329-346.

10. Blobel G, Sabatini DD: Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes. J Cell Biol 1970, 45:130-145. 11. Smith WP, Tai PC, Davis BD: Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis. Proc Natl Acad Sci U S A 1978, 75:5922-5925. 12. Bhushan S, Gartmann M, Halic M, Armache JP, Jarasch A, Mielke T, Berninghausen O, Wilson DN, Beckmann R: alphaHelical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat Struct Mol Biol 2010, 17:313-317. 13. Bhushan S, Meyer H, Starosta AL, Becker T, Mielke T, Berninghausen O, Sattler M, Wilson DN, Beckmann R: Structural basis for translational stalling by human cytomegalovirus (hCMV) and fungal arginine attenuator peptide (AAP). Mol Cell 2010, 40:138-146. 14. Kramer G, Ramachandiran V, Hardesty B: Cotranslational folding—omnia mea mecum porto? Int J Biochem Cell Biol 2001, 33:541-553. 15. Woolhead CA, McCormick PJ, Johnson AE: Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 2004, 116:725-736. 16. Kosolapov A, Tu L, Wang J, Deutsch C: Structure acquisition of the T1 domain of Kv1.3 during biogenesis. Neuron 2004, 44:295-307. 17. Lu J, Deutsch C: Folding zones inside the ribosomal exit tunnel. Nat Struct Mol Biol 2005, 12:1123-1129. www.sciencedirect.com

Ribosome stalling Wilson, Arenz and Beckmann 131

18. Lu J, Deutsch C: Secondary structure formation of a transmembrane segment in Kv channels. Biochemistry 2005, 44:8230-8243.

37. Ude S, Lassak J, Starosta AL, Kraxenberger T, Wilson DN, Jung K: Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science 2013, 339:82-85.

19. Kosolapov A, Deutsch C: Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol Biol 2009, 16:405-411.

38. Gutierrez E, Shin BS, Woolstenhulme CJ, Kim JR, Saini P, Buskirk AR, Dever TE: eIF5A promotes translation of polyproline motifs. Mol Cell 2013, 51:35-45.

20. Voss NR, Gerstein M, Steitz TA, Moore PB: The geometry of the ribosomal polypeptide exit tunnel. J Mol Biol 2006, 360:893-906. 21. Lu J, Deutsch C: Electrostatics in the ribosomal tunnel modulate chain elongation rates. J Mol Biol 2008, 384:73-86. 22. Evans MS, Sander IM, Clark PL: Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo. J Mol Biol 2008, 383:683-692. 23. Nilsson OB, Hedman R, Marino J, Wickles S, Bischoff L, Johansson M, Muller-Lucks A, Trovato F, Puglisi JD, O’Brien EP et al.: Cotranslational protein folding inside the ribosome exit tunnel. Cell Rep 2015, 12:1533-1540. 24. Wohlgemuth I, Brenner S, Beringer M, Rodnina MV: Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates. J Biol Chem 2008, 283:32229-32235. 25. Dimitrova LN, Kuroha K, Tatematsu T, Inada T: Nascent peptidedependent translation arrest leads to Not4p-mediated protein degradation by the proteasome. J Biol Chem 2009, 284: 10343-10352. 26. Chiabudini M, Tais A, Zhang Y, Hayashi S, Wolfle T, Fitzke E, Rospert S: Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae. Mol Cell Biol 2014, 34:4062-4076. 27. Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li GW, Zhou S, King D, Shen PS, Weibezahn J et al.: A ribosomebound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell 2012, 151:1042-1054. 28. Koutmou KS, Schuller AP, Brunelle JL, Radhakrishnan A, Djuranovic S, Green R: Ribosomes slide on lysine-encoding homopolymeric A stretches. Elife 2015:4. 29. Pavlov MY, Watts RE, Tan Z, Cornish VW, Ehrenberg M, Forster AC: Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proc Natl Acad Sci U S A 2009, 106:50-54.

39. Peil L, Starosta AL, Lassak J, Atkinson GC, Virumae K, Spitzer M, Tenson T, Jung K, Remme J, Wilson DN: Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P. Proc Natl Acad Sci U S A 2013, 110:15265-15270. 40. Woolstenhulme CJ, Guydosh NR, Green R, Buskirk AR: Highprecision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep 2015, 11:13-21. 41. Hersch SJ, Wang M, Zou SB, Moon KM, Foster LJ, Ibba M, Navarre WW: Divergent protein motifs direct elongation factor P-mediated translational regulation in Salmonella enterica and Escherichia coli. MBio 2013:4. 42. Elgamal S, Katz A, Hersch SJ, Newsom D, White P, Navarre WW, Ibba M: EF-P dependent pauses integrate proximal and distal signals during translation. PLoS Genet 2014, 10:e1004553. 43. Starosta AL, Lassak J, Peil L, Atkinson GC, Virumae K, Tenson T, Remme J, Jung K, Wilson DN: Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site. Nucleic Acids Res 2014, 42: 10711-10719. 44. Seefeldt AC, Nguyen F, Antunes S, Perebaskine N, Graf M, Arenz S, Inampudi KK, Douat C, Guichard G, Wilson DN et al.: The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex. Nat Struct Mol Biol 2015, 22:470-475. 45. Roy RN, Lomakin IB, Gagnon MG, Steitz TA: The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin. Nat Struct Mol Biol 2015, 22:466-469. 46. Vazquez-Laslop N, Mankin AS: Triggering peptide-dependent translation arrest by small molecules: ribosome stalling modulated by antibiotics. In Regulatory Nascent Polypeptides. Edited by Ito K. Springer; 2014:165-186. 47. Vazquez-Laslop N, Thum C, Mankin AS: Molecular mechanism of drug-dependent ribosome stalling. Mol Cell 2008, 30: 190-202.

30. Johansson M, Ieong KW, Trobro S, Strazewski P, Aqvist J, Pavlov MY, Ehrenberg M: pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the Asite aminoacyl-tRNA. Proc Natl Acad Sci U S A 2011, 108:79-84.

48. Ishii E, Chiba S, Hashimoto N, Kojima S, Homma M, Ito K, Akiyama Y, Mori H: Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria. Proc Natl Acad Sci U S A 2015, 112:E5513-E5522.

31. Muto H, Ito K: Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin. Biochem Biophys Res Commun 2008, 366:1043-1047.

49. Chiba S, Ito K: Multisite ribosomal stalling: a unique mode of  regulatory nascent chain action revealed for MifM. Mol Cell 2012. Report identifying multi-site ribosome stalling due to the polypeptide arrest sequence of MifM.

32. Doerfel LK, Wohlgemuth I, Kothe C, Peske F, Urlaub H,  Rodnina MV: EF-P is essential for rapid synthesis of proteins containing consecutive proline residues. Science 2013, 339: 85-88. Together with reference 37, these reports reveal that in bacteria translation stalling due to polyproline sequences is relieved by the translation elongation factor EF-P. 33. Hayes CS, Bose B, Sauer RT: Proline residues at the C terminus of nascent chains induce SsrA tagging during translation termination. J Biol Chem 2002, 277:33825-33832. 34. Tanner DR, Cariello DA, Woolstenhulme CJ, Broadbent MA, Buskirk AR: Genetic identification of nascent peptides that induce ribosome stalling. J Biol Chem 2009, 284: 34809-43818.

50. Tsai A, Kornberg G, Johansson M, Chen J, Puglisi JD: The dynamics of SecM-induced translational stalling. Cell Rep  2014. Single molecule study addressing the mechanism of SecM-mediated translational stalling and revealing multi-site stalling in SecM. 51. Ramu H, Vazquez-Laslop N, Klepacki D, Dai Q, Piccirilli J, Micura R, Mankin AS: Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center. Mol Cell 2011, 41: 321-330. 52. Spevak CC, Ivanov IP, Sachs MS: Sequence requirements for ribosome stalling by the arginine attenuator peptide. J Biol Chem 2010, 285:40933-40942.

35. Woolstenhulme CJ, Parajuli S, Healey DW, Valverde DP, Petersen EN, Starosta AL, Guydosh NR, Johnson WE, Wilson DN, Buskirk AR: Nascent peptides that block protein synthesis in bacteria. Proc Natl Acad Sci U S A 2013, 110:E878-E887.

53. Fang P, Spevak C, Wu C, Sachs M: A nascent polypeptide domain that can regulate translation elongation. Proc Natl Acad Sci U S A 2004, 101:4059-4064.

36. Ingolia NT, Lareau LF, Weissman JS: Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 2011, 147:789-802.

54. Matheisl S, Berninghausen O, Becker T, Beckmann R: Structure  of a human translation termination complex. Nucleic Acids Res 2015.

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Reports a cryo-EM structure of a human ribosome stalled during translation of the uORF of the cytomegalovirus gp48 gene, revealing a helix peptide in the tunnel that inhibits eRF1 action during termination by perturbation of the PTC.

72. Butkus ME, Prundeanu LB, Oliver DB: Translocon ‘‘pulling’’ of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation. J Bacteriol 2003, 185:6719-6722.

55. Chiba S, Kanamori T, Ueda T, Akiyama Y, Pogliano K, Ito K: Recruitment of a species-specific translational arrest module to monitor different cellular processes. Proc Natl Acad Sci U S A 2011, 108:6073-6078.

73. Gumbart J, Schreiner E, Wilson DN, Beckmann R, Schulten K: Mechanisms of SecM-mediated stalling in the ribosome. Biophys J 2012, 103:331-341.

56. Luo ZL, Sachs MS: Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa. J Bacteriol 1996, 178:2172-2177. 57. Gong F, Yanofsky C: Instruction of translating ribosome by nascent peptide. Science 2002, 297:1864-1867. 58. Lovett PS, Rogers EJ: Ribosome regulation by the nascent peptide. Microbiol Rev 1996, 60:366-385. 59. Ramu H, Mankin A, Vazquez-Laslop N: Programmed drugdependent ribosome stalling. Mol Microbiol 2009, 71:811-824. 60. Sohmen D, Chiba S, Shimokawa-Chiba N, Innis CA,  Berninghausen O, Beckmann R, Ito K, Wilson DN: Structure of the Bacillus subtilis 70S ribosome reveals the basis for speciesspecific stalling. Nat Commun 2015, 6:6941. Reports a cryo-EM structure of the Gram-positive Bacillus subtilis ribosome stalled during translation of the MifM arrest sequence, providing insights into species-specificity of ribosome stalling. 61. Bischoff L, Berninghausen O, Beckmann R: Molecular basis for  the ribosome functioning as an L-tryptophan sensor. Cell Rep 2014, 9:469-475. Reports a cryo-EM structure of the TnaC-stalled ribosome in the presence of L-tryptophan, revealing how TnaC and tryptophan cooperate to perturb the PTC and prevent RF2-mediated translation termination. 62. Arenz S, Meydan S, Starosta AL, Berninghausen O, Beckmann R,  Vazquez-Laslop N, Wilson DN: Drug sensing by the ribosome induces translational arrest via active site perturbation. Mol Cell 2014, 56:446-452. Reports a cryo-EM structure of the ErmCL-stalled ribosome in the presence of the antibiotic erythromycin, revealing how ErmCL and erythromycin cooperate to perturb the PTC and peptide bond formation during translation elongation. 63. Geballe AP, Leach FS, Mocarski ES: Regulation of cytomegalovirus late gene expression: gamma genes are controlled by posttranscriptional events. J Virol 1986, 57: 864-874. 64. Degnin CR, Schleiss MR, Cao J, Geballe AP: Translational inhibition mediated by a short upstream open reading frame in the human cytomegalovirus gpUL4 (gp48) transcript. J Virol 1993, 67:5514-5521. 65. Cao JH, Geballe AP: Coding sequence-dependent ribosomal arrest at termination of translation. Mol Cell Biol 1996, 16: 603-608. 66. Janzen DM, Frolova L, Geballe AP: Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA. Mol Cell Biol 2002, 22:8562-8570. 67. Alderete JP, Jarrahian S, Geballe AP: Translational effects of mutations and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene. J Virol 1999, 73:8330-8337. 68. Youngman EM, Brunelle JL, Kochaniak AB, Green R: The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 2004, 117:589-599. 69. Wei J, Wu C, Sachs MS: The arginine attenuator peptide interferes with the ribosome peptidyl transferase center. Mol Cell Biol 2012, 32:2396-2406.

74. Ismail N, Hedman R, Schiller N, von Heijne G: A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration. Nat Struct Mol Biol 2012, 19:1018-1022. Reports an elegant method to monitor the strength of the pulling force required to relieve nascent polypeptide-mediated translation arrest. 75. Chiba S, Ito K: MifM monitors total YidC activities of Bacillus subtilis including that of YidC2, the target of regulation. J Bacteriol 2014. 76. Gong F, Ito K, Nakamura Y, Yanofsky C: The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro). Proc Natl Acad Sci U S A 2001, 98:8997-9001. 77. Cruz-Vera L, Rajagopal S, Squires C, Yanofsky C: Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. Mol Cell 2005, 19:333-343. 78. Cruz-Vera LR, Yanofsky C: Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression. J Bacteriol 2008, 190:4791-4797. 79. Kannan K, Kanabar P, Schryer D, Florin T, Oh E, Bahroos N, Tenson T, Weissman JS, Mankin AS: The general mode of  translation inhibition by macrolide antibiotics. Proc Natl Acad Sci U S A 2014, 111:15958-15963. (Together with reference 81) Provides new insights into the mechanism of action of macrolide antibiotics, revealing that macrolides can induce translation arrest at specific tripeptide motifs within nascent polypeptide chains. 80. Kannan K, Vazquez-Laslop N, Mankin AS: Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel. Cell 2012, 151:508-520. 81. Davis AR, Gohara DW, Yap MN: Sequence selectivity of macrolide-induced translational attenuation. Proc Natl Acad Sci U S A 2014, 111:15379-15384. 82. Gupta P, Sothiselvam S, Vazquez-Laslop N, Mankin AS: Deregulation of translation due to post-transcriptional modification of rRNA explains why erm genes are inducible. Nat Commun 2013, 4:1984. 83. Johansson M, Chen J, Tsai A, Kornberg G, Puglisi JD: Sequencedependent elongation dynamics on macrolide-bound ribosomes. Cell Rep 2014. 84. Vazquez-Laslop N, Klepacki D, Mulhearn DC, Ramu H, Krasnykh O, Franzblau S, Mankin AS: Role of antibiotic ligand in nascent peptide-dependent ribosome stalling. Proc Natl Acad Sci U S A 2011, 108:10496-10501. 85. Noeske J, Huang J, Olivier NB, Giacobbe RA, Zambrowski M, Cate JH: Synergy of streptogramin antibiotics occurs independently of their effects on translation. Antimicrob Agents Chemother 2014, 58:5269-5279. 86. Schmeing TM, Seila AC, Hansen JL, Freeborn B, Soukup JK, Scaringe SA, Strobel SA, Moore PB, Steitz TA: A pretranslocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits. Nat Struct Biol 2002, 9:225-230.

70. Dalbey RE, Wang P, Kuhn A: Assembly of bacterial inner membrane proteins. Annu Rev Biochem 2011, 80:161-187.

87. Schmeing TM, Huang KS, Kitchen DE, Strobel SA, Steitz TA: Structural insights into the roles of water and the 20 hydroxyl of the P site tRNA in the peptidyl transferase reaction. Mol Cell 2005, 20:437-448.

71. Chiba S, Lamsa A, Pogliano K: A ribosome-nascent chain sensor of membrane protein biogenesis in Bacillus subtilis. EMBO J 2009, 28:3461-3475.

88. Schmeing TM, Huang KS, Strobel SA, Steitz TA: An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature 2005, 438:520-524.

Current Opinion in Structural Biology 2016, 37:123–133

www.sciencedirect.com

Ribosome stalling Wilson, Arenz and Beckmann 133

89. Doerfel LK, Wohlgemuth I, Kubyshkin V, Starosta AL, Wilson DN, Budisa N, Rodnina MV: Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome. J Am Chem Soc 2015, 137:12997-13006.

91. Behrmann E, Loerke J, Budkevich TV, Yamamoto K, Schmidt A, Penczek PA, Vos MR, Burger J, Mielke T, Scheerer P et al.: Structural snapshots of actively translating human ribosomes. Cell 2015, 161:845-857.

90. Sothiselvam S, Liu B, Han W, Ramu H, Klepacki D, Atkinson GC, Brauer A, Remm M, Tenson T, Schulten K et al.: Macrolide antibiotics allosterically predispose the ribosome for translation arrest. Proc Natl Acad Sci U S A 2014, 111:9804-9809.

92. Weixlbaumer A, Jin H, Neubauer C, Voorhees R, Petry S, Kelley A, Ramakrishnan V: Insights into translational termination from the structure of RF2 bound to the ribosome. Science 2008, 322:953-956.

www.sciencedirect.com

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