Co-translational mechanisms of protein maturation

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ScienceDirect Co-translational mechanisms of protein maturation Felix Gloge, Annemarie H Becker, Gu¨nter Kramer and Bernd Bukau Protein biogenesis integrates multiple finely regulated mechanisms, ensuring nascent polypeptide chains are correctly enzymatically processed, targeted to membranes and folded to native structure. Recent studies show that the cellular translation machinery serves as hub that coordinates the maturation events in space and time at various levels. The ribosome itself serves as docking site for a multitude of nascent chain-interacting factors. The movement of ribosomes along open reading frames is non-uniformous and includes pausing sites, which facilitates nascent chain folding and perhaps factor engagement. Here we summarize current knowledge and discuss emerging concepts underlying the critical interplay between translation and protein maturation in E. coli. Addresses Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany Corresponding authors: Kramer, Gu¨nter ([email protected]) and Bukau, Bernd ([email protected])

Current Opinion in Structural Biology 2014, 24:24–33 This review comes from a themed issue on Folding and binding Edited by James Bardwell and Gideon Schreiber

0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.sbi.2013.11.004

Introduction The maturation of newly synthesized polypeptides into correctly processed, translocated and natively folded proteins is intimately linked to protein synthesis. Folding steps frequently initiate during ongoing translation, albeit to higher degree in eukaryotes than in prokaryotes, possibly because in eukaryotes translation speed is lower and proteins are larger on average [1,2,3,4]. In several multi-domain proteins, co-translational folding facilitates acquisition of the native state [5–8]. Cotranslational folding is therefore considered a common strategy for increasing the efficiency of protein biogenesis. The underlying mechanisms, however, remain unclear and details may differ from protein to protein, dependent on the particular folding requirements and structural features. Nonetheless, coupling polypeptide synthesis with folding offers several general advantages. Firstly, vectorial folding, initiating with N-terminal parts of the nascent chain, will facilitate more ordered folding trajectories that prevent misfolding into nonfunctional conformers, especially in Current Opinion in Structural Biology 2014, 24:24–33

proteins with multiple domains [1]. Secondly, translation elongation is not uniform. The speed at which ribosomes move along mRNAs varies considerably [9,10]. Incorporation of slow phases may allow for the regulation of folding, membrane targeting and enzymatic processing. Thirdly, the exit area of the ribosomal tunnel offers a docking platform for the coordinated binding of multiple different nascent chain-interacting proteins which promote distinct maturation steps [11–16]. Fourthly, early acquisition of secondary and tertiary structure [17–20] may shorten exposure of newly synthesized polypeptides to the cellular environment whilst in the non-native state. Recent analyses reveal critical steps of the co-translational protein maturation events in E. coli, and establish a conceptual framework for the mechanisms governing protein biogenesis.

Ribosomal influence on early nascent chain folding Nascent polypeptides traverse the 50S subunit from the peptidyl transferase center to the ribosome surface via a peptide exit tunnel extending 80–100 A˚ [21,22] (Figure 1a). The tunnel can accomodate a nascent chain of approximately 30–35 amino acids in extended conformation [23,24]. Originally, interactions between the growing polypeptide and the ribosome were thought to be counter-productive, with a ‘Teflon-like’ interior ascribed to the tunnel to avoid these [21,22]. However, it is an emerging concept that the tunnel plays an active role in protein folding and interacts with the transiting nascent chain to regulate translation elongation (discussed further below). Specific folding zones at the distal end of the tunnel, beyond the constriction point formed by ribosomal proteins L4 and L22 allow formation of a-helices and b-hairpins [17,23,25–27]. Theoretical and crosslinking studies further indicate an early folding environment for initial probing for low entropy or simple secondary protein structures, close to the funnel-shaped exit vestibule of the ribosome [18,28]. On the other hand, at the ribosomal surface formation of complex folds and native-like structures of nascent chains is inhibited. Recent experimental and computational data show that nascent chain dynamics is restricted in proximity of the ribosome which thereby delays folding [28–31]. By providing a folding zone for secondary structure elements and local tertiary structures while simultaneously preventing distant (potentially detrimental) interactions, the ribosome may reduce the conformational space explored by the nascent chain, effectively channeling the conformational search towards the native fold. www.sciencedirect.com

Co-translational mechanisms of protein maturation Gloge et al. 25

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Mechanisms of translation attenuation. (a) Cross-section of the 50S ribosomal subunit highlighting the peptide exit tunnel which connects the PTC with the ribosome surface. The constriction point, consisting of L4 and L22, is colored in blue. The part of the tunnel wall formed by L23 is colored in red. (b) Translation speed can be reduced by several mechanisms involving mRNA features including rare codon clusters, secondary structure elements and Shine-Dalgarno like sequences, or polybasic stretches in nascent chains that interact with the negatively charged wall of the ribosomal exit tunnel.

Fine-tuning translation speed affects nascent chain folding Compared to in vitro folding of proteins, which for most proteins occurs on a millisecond to second timescale [32], translation is rather a slow process with elongation rates of 10–20 amino acids/s in prokaryotes [33]. The pace by which nascent polypeptides emerge from the tunnel exit therefore limits the folding rate of most proteins. Initial protein domains may therefore acquire secondary/tertiary structure before subsequent domains emerge, precluding potential interference with the folding process. Domainwise folding may also explain the observation that cotranslational folding is frequently more efficient than refolding of the same full-length denatured protein chain [1,34]. Accordingly, slowing down translation often enhances folding efficiency in vivo, while fast translation increases the total amount of protein synthesized but also the fraction of misfolded proteins [35–39]. In this context it is significant that speed of translation is not uniform along the length of an mRNA [9,10,40]. Translation speed may therefore directly control and fine-tune protein folding, becoming especially relevant for large or multi-domain proteins [1,34,41,42]. Generally applied, this principle of regulation would imply that the genetic code not only dictates amino acid chain sequence, but also influences the folding of proteins into the native state. Several mechanisms exist to slow down translation speed or cause translational pausing (Figure 1b): (1) incorporation of ‘rare’ codons for which complementary charged tRNAs are scarce; (2) basepairing of stretches in the www.sciencedirect.com

ribosomal 16S rRNA with Shine-Dalgarno-like sequences in mRNAs; (3) secondary structure elements in mRNAs which provide a physical block for the ribosome; (4) physical interaction of nascent polypeptides with the ribosomal exit tunnel. The relative impact of these mechanisms on translation control is not yet entirely clear, although existing data directly support a role for rare codons in the control of protein folding. Rare codons are not equally distributed within mRNA sequences [43–45] and often cluster downstream of inter-domain borders [46,47] and in areas encoding structural elements that form co-translationally, including a-helices (Figure 1b1) [48]. This has led to speculation that an evolutionarily conserved mechanism facilitates co-translational folding. Accordingly, synonymous mutations in human MDR1 gene change the folding and conformation of the MDR1 gene product [49] and non-optimal codon usage in the FRQ gene of Neurospora affects function, expression level and phosphorylation pattern of FRQ protein [50] implying altered protein structure. Furthermore, Zhang et al. report that either replacement of rare codons in sufI or over-expression of rare tRNAs [45] is detrimental to correct folding of the multi-domain protein SufI in E. coli. However, recent ribosome profiling studies in E. coli have challenged the relevance of rare codons in translational pausing. Instead, frequently occurring Shine-Dalgarnolike sequences in mRNA coding sequences are suggested to be strong attenuators of translation elongation Current Opinion in Structural Biology 2014, 24:24–33

26 Folding and binding

(Figure 1b2) [51]. Shine-Dalgarno sequences are located 8–11 nucleotides upstream of the AUG start-codon and are required for translation initiation by basepairing with complementary anti-Shine-Dalgarno sequences within the 16S rRNA of the 30S ribosomal subunit. Shine-Dalgarno-like sequences within genes may be implicated in the promotion of translational frameshifts, transcriptional regulation and potentially also the modulation of cotranslational folding [51]. The positioning of these Shine-Dalgarno-like sequences in conserved open reading frames (ORFs) is also preserved across prokaryotic species, further suggesting functional relevance. Direct evidence for a specific role of these motifs in protein folding is, however, lacking so far. Secondary structure elements in mRNAs provide a third way of slowing down translation (Figure 1b3). Folded structures such as stem-loops constitute kinetic barriers for translating ribosomes [52,53] and are employed by cells for programmed frameshifting [54,55] and protein expression level control [56]. An involvement of mRNA secondary structures in the control of co-translational protein folding has been suggested [57] but the relevance of this mechanism is not well explored. Translational speed is also regulated by direct interactions of nascent chains with the ribosomal exit tunnel (Figure 1b4). The tunnel has strong electronegative character and electrostatic interaction of polybasic stretches in nascent proteins with the tunnel wall reduces the rate of chain elongation [58–60]. Interestingly, signal sequences of translocated proteins do feature an N-terminal stretch of positively charged amino acids [61], consistent with a potential role in slowing down translation until the nascent chain can be channeled into the respective translocation pathway. A further mechanism for translational pausing relies on interactions of some nascent chains (such as SecM) with the ribosomal tunnel, via specific ribosome arrest peptides (RAPs) [62]. RAPs employ the tunnel constriction point and other structural features to communicate a transient stop-signal to the peptidyl-transferase center of the ribosome. Signal recognition and transfer apparently occurs via tightly orchestrated structural relay cascades involving both rRNA and ribosomal proteins. Although a specific role for RAPs in protein maturation has not been demonstrated, these highly specific peptide sequences are striking examples of the finely tuned communication between nascent chains and ribosomes.

The tunnel exit as docking site for nascent chain-interacting factors The tunnel exit is encircled by a proteinaceous ring formed by ribosomal proteins L17, L22, L32, L24, L29 and L23 (Figure 2a) [21,22]. Among these proteins L23 is unique because it contributes to the inner wall of the Current Opinion in Structural Biology 2014, 24:24–33

tunnel and functions as the central docking site for multiple nascent chain-interacting factors, including the trigger factor (TF) chaperone, the signal recognition particle (SRP) and the SecA ATPase [11,15,63–65] (Figure 3b). These features of L23 apparently enable the ribosome to both sense nascent polypeptides inside the tunnel and relay this information to the ribosomal surface, by unknown mechanism. For the SRP, this relay via L23 results in rapid recruitment of the particle to translating ribosomes [66,67]. In E. coli, SRP recruitment occurs independently of the presence of an a-helical signal anchor sequence (SAS). In contrast, yeast ribosomes distinguish between nascent chains and recruit SRP selectively, to ribosome-nascent chain complexes (RNCs) carrying an N-terminal signal sequence [68]. Whether L23-mediated signaling is used for the recruitment of other factors is unclear, but a recent publication suggests that the ribosome may reduce the recruitment of TF in response to the presence of a-helical segments or signal sequences of nascent chains in distal segments of the exit tunnel [69]. The tunnel exit area provides further binding sites for nascent chain-interacting factors (Figure 2b). SRP associates with L23 and L29, with charged interactions between Ffh, the protein moiety of SRP, and L23 making major contributions [63,65]. Recognition of a signal anchor sequence in the nascent chain results in additional contacts with rRNA, L24, L22 and L18. Peptide deformylase (PDF), which removes the formyl group of the Nterminal methionine of nascent chains, associates with L22 and rRNA via a conserved, positively charged Cterminal helix [14] (Figure 2c). L22 also participates in forming the constriction point of the tunnel interior, raising the speculation of a coordinated recruitment of PDF, depending on the translational state of the ribosome. Methionine aminopeptidase (MAP), which hydrolyses the N-terminal methionine of approximately 70% of all nascent chains, employs a positively charged structured loop that binds an area between L23 and L17, presumably ribosomal RNA [13] (Figure 2c). Although this charged loop is present only in the eubacterial MAPtype 1 protein subfamily, eukaryotic MAP family members have insertions that are different in sequence and size, but similarly positively charged (Figure 2d). We propose these insertion sequences also serve as ribosome binding sites in eukaryotic MAPs. Ribosome association of nascent chain-interacting factors appears to involve the common underlying theme of electrostatic interactions (Figure 2c). Presumably this also helps interacting factors to establish initial spatial proximity to the strongly electro-negative ribosomes, increasing local concentrations of these factors. Another common theme relates to the strength with which the various nascent chain-interacting factors interact with specific binding sites at the tunnel exit. None of the proteins www.sciencedirect.com

Co-translational mechanisms of protein maturation Gloge et al. 27

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Overview of the ribosomal proteins near the peptide exit channel and nascent chain-interacting factors binding to this region. (a) Top view of the large ribosomal subunit and the relevant proteins surrounding the tunnel exit (L17, blue; L32, yellow; L22, purple; L29, orange; L24, black; L23, red). (b) Footprints of ribosome associated nascent chain-interacting factors in E. coli. Shown are the footprints of PDF (yellow), SRP (green), MAP (black) and TF (blue). (c) Positioning of PDF, MAP, TF and SRP on the ribosome surface [13,14,62,63]. The electrostatic surface potentials of the proteins are calculated via APBS [113] and visualized by showing the isopotential contours at values of 1 kTe 1 (red) and + 1 kTe 1 (blue). Ribosomal proteins close to the binding site are colored in orange. (d) Left: electrostatic surface potentials of MAPs from E. coli (2MAT), P. furiosus (1XGM), H. sapiens type 1 MAP (2EA2) and H. sapiens type 2 MAP (1R5G). Right: alignment of E. coli type 1 MAP and P. furiosus type 2 MAP reveals that in type 2 MAPs, a positively charged helical domain (purple) is inserted at the position of the type 2 ribosome binding loop (green).

bind stably to the ribosome. Instead, kinetics are usually fast, ranging from seconds for TF (t1/2 = 10–15 s) to as short as milliseconds for SRP during initial ribosome (t1/2 = 69 ms), and PDF (t1/2 = 3 ms) binding [13,66,70,71]. Rapid kinetics are a prerequisite for all www.sciencedirect.com

processes to occur within a time-frame pertinent to translation. Some of these factors have interfering or even mutually exclusive ribosomal binding sites (Figure 2b). For instance, E. coli PDF and MAP, two enzymes which must act consecutively on nascent chains, exclude each Current Opinion in Structural Biology 2014, 24:24–33

28 Folding and binding

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Factors mediating N-terminal enzymatic processing, folding and membrane targeting of proteins in E. coli. (a) Co-translational targeting of nascent chains carrying a SAS to the translocon by SRP. (b) Co-translational recognition of proteins destined for translocation across the cytoplasmic membrane by SecA for targeting into the post-translational translocation mode. (c) Nascent cytoplasmic proteins are N-terminally processed by PDF and MAP. Following interaction with TF proteins either fold spontaneously or require further assistance by the DnaK/DnaJ/GrpE and GroEL/GroES chaperone system acting downstream.

other from binding to the ribosome, but show very rapid binding/release kinetics [13].

Process coordination at the ribosome The engagement of the different nascent chain-interacting factors requires precise orchestration, to ensure accurate coordination of folding, enzymatic processing and membrane targeting. The emerging picture is that these factors act as separate entities, rather than as preassembled protein complexes. Ribosome binding of these factors in E. coli even exhibits negative interference (PDF versus TF) or is mutually exclusive (PDF versus MAP). Although the transient ribosome binding kinetics of the involved factors may in principle permit some stochasticity in the sequence of processing events, a more stringent regulation imposing a specific order of events seems to occur. For instance N-terminal methionine excision by Current Opinion in Structural Biology 2014, 24:24–33

MAP is hampered by TF binding and impossible for some newly synthesized proteins once they have attained native structure [13]. Ribosome association of PDF and MAP is therefore important for efficient processing, as it ensures both enzymes act on short nascent chains of 44– 48 residues [13] before native folding and TF interaction take place. PDF and MAP thus are among the first factors a nascent chain encounters outside the tunnel. Deformylation and N-terminal methionine excision must also be coordinated with the targeting of proteins to the translocon, involving SRP, SecA and SecB. In prokaryotes, ribosomes synthesizing inner membrane proteins are co-translationally targeted to the SecY translocon by the SRP [72,73] (Figure 3a). SRP recognizes peptides resembling the SAS of translocation substrates via a hydrophobic groove that is tailored to bind www.sciencedirect.com

Co-translational mechanisms of protein maturation Gloge et al. 29

N-terminal signal anchor sequences with a kD of 1.5  0.4 mM [74]. On the basis of crosslinking studies SRP is estimated to associate with nascent substrates of a minimal length of approx. 37 residues [75]. In vitro kinetic measurements using fluorescently labeled SRP reveal that SRP binds to the ribosome in a multistep mode, involving at least three kinetically distinct steps [66]: (1) scanning mode, allowing for fast initial sampling of vacant ribosomes (kD = 60 nM; koff = 10 s 1); (2) standby mode, established upon encountering ribosomes translating SAS-containing short peptides of 35 amino acids (kD = 3.5 nM, koff = 1 s 1). Formation of this intermediate complex may be facilitated by allosteric signaling via L23 which senses nascent chains inside the tunnel; (3) targeting mode, characterized by high affinity binding of SRP to ribosome-nascent chain complexes upon recognition of the SAS (kD = 2 nM; koff = 0.08 s 1). This step facilitates binding of the SRP receptor to the RNC–SRP targeting complex. Then, controlled by GTP hydrolysis, SRP undergoes a large conformational change which repositions its NG-domain in relation to the RNA part, to prepare the complex for docking and transfer of the nascent substrate to the translocon [76,77]. This order of events establishes a multistep control process allowing SRP to faithfully select proteins destined for co-translational translocation and grants SRP privileged access to nascent chains right when they emerge at the tunnel exit. Interestingly, SRP and PDF do not bind competitively to ribosomes [13], which is in line with the virtually complete deformylation of the entire E. coli proteome including SRP substrates [78,79]. However, the footprints of SRP and PDF bound separately to the ribosome predict a steric clash upon simultaneous binding of both factors (Figure 2b). Efficient deformylation may be explained by either flexible positioning of SRP or the high dynamics of binding of PDF and SRP, before the SAS is fully exposed at the tunnel exit. Concerning the interplay between MAP and SRP, data are not available. However, we note that competitive binding of both proteins to the ribosome would have no consequences since most signal sequences contain a charged stretch of amino acids succeeding the N-terminal methionine, which exempts SRP substrates from processing by MAP [61,80]. Proteins of the periplasm and the outer membrane on the other hand are targeted to the translocon via the SecA– SecB pathway [81,82] (Figure 3b). Until recently, SecA dependent translocation across the cytoplasmic membrane was considered an exclusively post-translational process. A recent report now shows that SecA crosslinks to L23 and engages exported proteins during synthesis [15]. Mechanistic insight into how ribosome binding of SecA is regulated is currently lacking, but this finding suggests that SecA recruits nascent substrates at the ribosome, creating a direct channel into the posttranslational translocation pathway. www.sciencedirect.com

Most cytosolic proteins engage the chaperone TF for folding [10] (Figure 3c). TF was initially proposed to associate with ribosomes already before the onset of translation, allowing nascent chains to emerge into the binding cavity of TF that arches over the tunnel exit [64,70,71]. However, recent findings challenge this proposal. First, the presence of TF in an in vitro transcription/ translation system decreases the affinity of PDF for ribosomes and the efficiency of N-terminal processing of nascent chains by PDF and MAP [13]. Second, profiling of translating ribosomes bound to TF shows that in vivo, TF associates selectively with translating ribosomes that harbor nascent chains with a length of at least 100 residues, and not indiscriminately with all ribosomes [10]. TF binding to ribosomes is therefore a rather late event during nascent chain maturation. This generates a prior kinetic window for the action of processing enzymes and SRP. The basis for the late TF association with ribosomes and nascent chains is, however, currently unclear.

Chaperone action in co-translational protein folding The folding of cytosolic proteins to the native state is assisted by a network of cooperating chaperones, with the main players in E. coli being TF and the ATP-dependent DnaK (with DnaJ and GrpE co-chaperones; KJE) and GroEL (with GroES co-chaperone; GroELS) systems [3,83–85]. Among these only TF is known to utilize a ribosome docking site for nascent chain engagement, placing TF at the beginning of a chaperone cascade [11,86,87]. Although these chaperones have completely different structures and mechanisms, they exhibit functional redundancy as indicated by genetic data. Only GroELS is strictly essential for cell viability, presumably because it is unconditionally required for the folding of a subset of approx. 85 proteins enriched in proteins with (ba)8 triosephosphate isomerase (TIM) barrel domains [88]. TF is dispensable for growth and DnaK is dispensable at physiological temperatures between approx. 20 and 378C. However, there is synthetic lethality of Dtig DdnaK mutants at temperatures above 308C accompanied by massive protein aggregation, which can be complemented by the overproduction of either GroELS or SecB [89–91]. Most likely, the absence of TF can be compensated by the activities of the other chaperones which prevent aggregation of misfolded proteins as holdases or rescue kinetically trapped conformers as foldases. The degree by which chaperone assistance is needed varies from protein to protein, and may be lowest for single domain proteins with simple folding pathways. Interactome studies indicate that at least 250 cytosolic protein species (6%) interact with GroEL during de novo synthesis, 20% with DnaK and almost all with TF [10,83,92]. These quantifications have to be taken as estimates, since chaperone–substrate interactions are Current Opinion in Structural Biology 2014, 24:24–33

30 Folding and binding

transient in nature and may be lost or gained during purification procedures. TF, the first chaperone that contacts nascent chains in E. coli [11,86,87], uses for substrate binding a cavity that extends over the entire length of the elongated TF molecule and is flanked by two helical arms [64,93]. The cavity is enriched in hydrophobic residues but also contains charged residues, allowing TF to accommodate a wide spectrum of nascent proteins over a fairly long stretch of the polypeptide chain. The interaction of TF with nascent chains is transient (t1/2 of the complex up to approx. 1 min) but can exceed the time TF is bound to the ribosome, implying it can stay associated with nascent chains during ongoing synthesis [70,71,94]. TF stabilizes unfolded and partially folded states of polypeptides, thereby acting in succession of the initial folding delay exerted by the ribosome [31,95,96] and protecting the nascent chains from proteolytic attack [97,98]. While these features are consistent with a role of TF as holdase, recent findings using single molecule approaches indicate a more active role as foldase [99]. TF facilitates local contacts and prevents distant interactions, and deepens energy valleys in the folding landscape. By promoting local folds and shielding this region from non-productive interactions with distant regions, TF allows domains to fold quasi-independently from the rest of the molecule. Moreover, polypeptides can even be unfolded by TF as long as they are ribosomeassociated and thermodynamically not too stable [31]. This may be a mechanism to rescue prematurely folded or misfolded protein species generated co-translationally. So far, these features of TF were identified using a small set of model substrates. It will be important to determine which cellular proteins require an active role of TF for folding and how the activity of TF relates to the discontinuous translation process. DnaK and DnaJ also associate with nascent polypeptides, but in addition act post-translationally. TF and DnaK/ DnaJ have similar specificities for peptides enriched in hydrophobic/aromatic and basic residues [100,101] but TF via its ribosome docking site competes out DnaK/ DnaJ for binding to nascent chains [102–104]. DnaK functions as a central hub within the chaperone network for newly synthesized proteins, since proteins accumulate significantly on DnaK if TF or GroEL are depleted. Therefore the DnaK system forms the functional interconnection between TF and GroELS [105]. A number of findings suggest that GroELS acts posttranslationally promoting the last assisted step in protein folding [106,107]. Firstly, the working principle of GroELS relies on the complete enclosure of the polypeptide substrate in a folding cavity formed by the double ring of GroEL 14mers and the GroES 7mer which acts as ATP-controlled lid [108,109]. In this Anfinsen cage, GroEL catalyzes the rescue of kinetically trapped states Current Opinion in Structural Biology 2014, 24:24–33

in protein folding, in some cases through imposed unfolding, and provides a confined folding space which channels substrates to the native state in an environment free of aggregation [110,111]. Secondly, GroEL recognizes global features of non-native proteins exhibiting increased surface hydrophobicity but significant tertiary structure, which qualifies late folding intermediates as typical GroELS substrates. Thirdly, substrates can be transferred from DnaK and TF to GroEL, placing GroEL downstream of a chaperone cascade. However, substrates can shuttle bidirectionally between DnaK and GroEL [112]. This suggests that with respect to substrate flux through the system, GroELS and KJE form a flexible bi-directional, rather than strictly uni-directional chaperone network in which, however, KJE may act first in the majority of the cases.

Conclusions An intricate system of maturation factors monitors and guides nascent polypeptide chains into functional protein structures. Accumulating evidence indicates the ribosome controls the central steps of this process. The ribosome monitors both length and secondary structural features of the nascent polypeptide, as well as features intrinsic to the mRNA molecule beyond the sequence code. These structural signals allow the detecting ribosome to coordinate membrane targeting, co-translational folding and enzymatic processing of nascent chains. The precise molecular mechanisms permitting this coordination, however, are not well explored and many fundamental questions remain. Precisely how do ribosomes sense hydrophobicity, secondary structure and length of nascent chains? How are the different signals integrated by a ribosome and translocated to the ribosomal surface? Is there a retrograde transfer of information from exposed parts of the nascent chain to the peptidyl transferase center for controlling translation speed dependent on nascent chain interactions? Is translation control further coordinated with the assembly of homomeric and oligomeric protein complexes? In this context, it will be especially challenging to untangle the coordination of processing and maturation steps in eukaryotic organisms, which contain additional ribosome associated factors, for example the ribosome-associated complex (RAC) and the nascent-chain associated complex (NAC), in addition to a diverse family of N-acetyltransferases and other enzymes that act co-translationally to modify nascent chains. Addressing these fundamental questions will require a combination of a wide variety of experimental tools and approaches including ribosome profiling, structural biology, biochemistry, computational modeling as well as theoretical analyses.

Acknowledgements We thank Lys Guilbride for critical contribution and editing of the manuscript. This work was supported by Collaborative Research Center SFB638, FOR967 and FOR1805 grants from the Deutsche Forschungsgemeinschaft to G.K. and B.B. www.sciencedirect.com

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2.

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