Modification, folding and targeting of nascent polypeptides

The tunnel exit site: During translation the growing nascent polypeptide passes through the exit tunnel and emerges at the solvent side, where it undergoes processing and early folding. The exit site is surrounded by rRNA, e.g. by the expansion segment ES27L, as well as by several ribosomal proteins. Four of these, uL22, uL23, uL24 and uL29, are conserved whereas eL19, eL31 and eL39 are not found in the prokaryotic ribosome (Fig. 4C/D) (YUSOPOVA & YUSOPOV, 2014). These variations are associated with the different N-termini processing of nascent chains in bacteria and eukaryotes: In prokaryotes, the nascent polypeptide contains a formyl group at the N-terminus which is due to the special formylation-modification of the aminoacylated initiator tRNA. As the eukaryotic initiator tRNA is not formylated, the positions corresponding to the bacterial bL17 and bL32 are occupied by the non-homologous protein eL31 (Fig. 4D) that serves as a global docking site for several non-ribosomal factors (MELNICOV et al., 2012).

The ribosome and especially the region of the tunnel exit serves as a platform for the spatially and temporally regulated association of targeting factors, enzymes or chaperones that act on the nascent polypeptide as it emerges from the tunnel exit (Fig. 5). Thus, the translation machinery provides opportunities to coordinate the synthesis of a polypeptide with its targeting or folding process (KRAMER et al., 2009). Amongst the early acting proteins are targeting factors like the signal recognition particle (SRP), and proteins that chemically modify the nascent chain like methionine aminopeptidases (MAPs), peptide deformylases (PDFs) or N-acetyl transferases (NATs). Chaperones like the bacterial Trigger Factor (TF) or the eukaryotic ribosome-associated factors NAC (nascent polypeptide-associated complex) and Ssb-RAC (ribosome-associated complex) guide initial protein folding. The ribosome quality control complex (RQC) and the Ccr4-Not complex are involved in mRNA surveillance as well as nascent chain ubiquitination and degradation (Fig. 5).

Nascent chain modifications: Many cellular proteins are subjected to chemical modifications some of which occur already during protein biosynthesis at the ribosomal tunnel exit. Among the factors that interact with nascent chains are enzymes that are involved in the N-terminal deformylation and methionine excision or in the enzymatic modification of the nascent chain by acetylation. Such modification is thought to influence the half-life of a protein as well as its ability to interact with other factors; thus, modification affects both the function and stability of a protein (KRAMER et al., 2009).

Methionine aminopeptidases that remove the N-terminal methionine of 30-50 % of nascent chains are ubiquitous and essential in all kingdoms (GIGLIONE et al., 2004). One homologue is found in bacteria and two in eukaryotes. MAPs can act co-translationally and bind directly to the ribosome (VETRO &CHANG, 2002) but need a minimal size of the nascent polypeptide of 40 aa for full functionality (BALL & KÄSBERG, 1973). At the ribosome MAPs bind via a positively charged loop to bL17 and uL23 which is in close proximity to the tunnel

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exit (SANDIKCI et al., 2013). In bacteria or mitochondria peptide deformylases (PDF) have to initially remove the formyl moiety from the N-terminal methionine before MAPs can act (PINE, 1969). PDFs associate with the ribosome via a C-terminal helix that binds to a groove between uL22 and bL32 (BINGEL-ERLENMEYER

et al., 2008). They have fast association and dissociation kinetics and compete with MAPs for ribosome binding (SANDIKCIet al., 2013). Simultaneous binding of MAP and PDF with the targeting factor SRP is possible in bacteria, indicating co-translational processing and targeting of nascent chains. In contrast, premature recruitment of the chaperone Trigger Factor or early polypeptide folding negatively affects the processing efficiency (SANDIKCI et al., 2013).

N-terminal acetylation is another modification that occurs co-translationally in approximately 80-90 % of mammalian cytosolic proteins, in 50 % of yeast proteins and only occasionally in prokaryotes. Several studies provide evidence for N-acetyl transferase activity, however the biological relevance of this modification is still unclear (POLEVODA &SHERMAN, 2003). Eukaryotes possess at least five different types of NATs, some of which acetylate the N-terminal methionine whereas others rely on the previous activity of MAPs. In yeast the N-acetyltransferase NatA could be crosslinked to nascent polypeptides, indicating its localization close to the ribosomal tunnel exit (GAUTSCHI et al., 2003) and pulldown experiments suggest NatA binding to the ribosomal proteins uL23 and uL29 (POLEVODA et al., 2008).

Figure 5: Co-translational processes on the nascent chain. Ribosome-associated factors interact with the nascent polypeptide and initiate transport to desired destinations, protein modification and/or folding by chaperones. Quality control factors prevent the accumulation of aberrant mRNAs and misfolded proteins. * deformylation and Trigger Factor (TF) are restricted to prokaryotes; # myristoylation, ubiquitination and the presence of the RQC and the Ccr4-Not complex are restricted to eukaryotes; + Ssb is specific to fungi.

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After methionine excision 1-4 % of eukaryotic proteins are subjected to co-translational myristoylation, which is the covalent attachment of a myristic acid moiety to an N-terminal glycine residue. It still remains elusive how myristoylation and translation are coupled (MEINNEL &GIGLIONE, 2008) and which function this modification provides. Proteins carrying a myristic acid modification are mostly targeted to lipid membranes where they are thought to play a role in the cellular communication network (GIGLIONE et al., 2015).

Nascent chain targeting by SRP: The signal recognition particle SRP is an abundant and universally conserved cytosolic ribonucleoprotein that co-translationally recognizes proteins containing a signal sequences (ss) or a transmembrane domain. These secretory or transmembrane proteins are targeted by SRP to the inner membrane of bacteria or to the endoplasmic reticulum (ER) in eukaryotes. Upon recognition of an N-terminal hydrophobic signal sequence, SRP binds to the ribosome nascent chain complex (RNC) and targets it to the membrane integrated SRP receptor SR (FtsY in bacteria). After transferring the RNC to the Sec61 translocation channel in the ER membrane (SecYEG in bacteria), the SRP-receptor complex dissociates. This cycle of protein targeting is regulated by guanosine triphosphatases (GTPases) present in SRP and its receptor, and GTP is hydrolyzed in a shared active site. The composition of SRP and its receptor varies strongly between kingdoms, although a combination of proteins and RNA is characteristic. In eukaryotes SRP consists of six proteins (SRP9, 14, 68, 72, 19 and 54) and the 7SL RNA with GTPase activity. The prokaryotic SRP is much less complex and composed of the 4.5S RNA and one protein with GTPase activity (Ffh = Fifty-four homologue) (GRUDNIK et al., 2009). The structure of eukaryotic SRP can be divided into an Alu- and a S-domain. The Alu-domain contains the SRP9/14 heterodimer and is involved in the elongation arrest of translation. The S-domain is composed of the remaining proteins and provides ss-binding and receptor interaction (HALIC et al., 2004). The overall composition of yeast SRP is similar to that of higher eukaryotes but with notable differences: it is characterized by the functional replacement of SRP9/14 by an SRP14 homodimer and the presence of a yeast-specific protein, SRP21, which is structurally related to SRP9. Furthermore, the yeast SRP RNA possesses a specific structure due to frequent insertions (STRUB et al., 1999), and the yeast homologue of SRP19 (Sec65) is a much larger protein (HANN &

WALTER, 1991).

SRP recognizes signal seqeunces that vary in their composition and length but share the common feature of a hydrophobic core region (VON HEIJNE, 1990). Remarkably, short nascent chains that are still enclosed within the ribosomal tunnel are sufficient for recruiting bacterial SRP, that possesses now a ~100-fold increased affinity for the translation machinery (BORNEMANN et al., 2008). This indicates a communication of tunnel wall proteins with those of the exit site. The transfer of this signal from the inside of the tunnel to the ribosomal surface occurs via a loop of the exit site protein uL23 that reaches into the tunnel (BORNEMANN et al., 2008). A similar mechanism could be observed in yeast, where SRP shows increased affinity for RNCs harboring nascent chains in the exit tunnel that contain a membrane anchor sequence (BERNDT et al., 2009). SRP contacts the ribosome via SRP54 and parts of its RNA and binds to the exit site proteins uL23 and uL29 (CROSS et al., 2009). Both, uL23 and either uL29 (HALIC et al., 2006; BECKER et al., 2009) or eL29 (VOORHEES et al., 2014) are also involved in the docking of RNCs to the translocon pore Sec61.

SRP binding to the ribosome induces arrest of translation elongation regulated via SRP9/14 and the RNA part of the Alu domain (THOMAS et al., 1997); a feature that is not found in bacteria. Upon translational arrest SRP delivers the RNC to the Sec61 translocon by interaction with its receptor in the ER membrane (HALIC

et al., 2006). The RNC engages the Sec61 translocon, which leads to resumption of translation, enabling the

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nascent chain to be directly transported into the ER lumen where it can fold into its final conformation. During translocation enzymes such as oligosaccharyl transferases (OST) and signal peptidases (SPase) can associate with the translocon and either N-glycosylate or cleave the signal peptide from the translocating nascent chain (NYATHI et al., 2013).

Although the exact interplay and spatial-temporal coordination of SRP and other ribosome-associated factors is not fully understood, studies in bacteria suggest the following order: SRP is the first factor interacting with the nascent chain (! 25 aa) followed by PDF and MAP (! 44 aa) which exclude each other from binding. Lastly TF binds nascent chains with a length of ~100 aa (SANDIKCI et al., 2013). In eukaryotes, where the composition of ribosome-associated factors is considerably more complex, further analyses are necessary to better understand the binding modes of all factors at the ribosome. A recent study analyzing binding of different factors to the universal adaptor site at the tunnel exit suggests that in the absence of NAC MAPs and SRP antagonize each other, proposing a role of NAC in regulating the access of MAP and SRP to the ribosome (NYATHI &POOL, 2015).

For the sake of completeness it should be mentioned that targeting in bacteria is more complex, as mainly membraneproteins are targeted co-translationally via SRP whereas periplasmic, outer membrane or secretory proteins that need to be translocated through the inner membrane into the periplasm are targeted via a late or post-translational pathway involving a set of Sec proteins (RAPOPORT, 2007).

De novo protein folding: Folding of the nascent polypeptide already starts within the ribosomal exit tunnel in which alpha-helical structures can form before and after the tunnel constriction (BHUSHAN et al., 2010). The formation of tertiary structures such as beta-hairpins has also been observed near the exit 80 Å away from the PTC where the tunnel opens gradually (KOSOLAPOV &DEUTSCH, 2009).

One mRNA transcript may be translated by several ribosomes simultaneously (so-called polysomes), where the ribosomes are arranged in a staggered or pseudo-helical organization around the mRNA with the tunnel exit sites facing outward (BRANDT et al., 2009). This arrangement maximizes the distances between the different nascent polypeptides emerging from neighboring ribosomes, preventing unfavorable interactions between them.

Different sets of ribosome-associated chaperones like TF in bacteria or Ssb-RAC in yeast interact early with the emerging nascent chain to prevent unspecific interactions or misfolding and aggregation (WEGRZYN &

DEUERLING, 2005). The principle of de novo protein folding is closer illuminated in a separate chapter (1.3.2) and the different systems of ribosome-associated chaperones are described in chapter 1.4.3.