Termination

The presence of one of three stop codons within the A-site terminates protein synthesis by releasing the nascent chain from the ribosome. These three stop codons are encoded as UAG, UAA and UGA. In contrary to canonical codons, stop codons are recognized by Class I release factors that mediate the hydrolysis of the ester bond of the peptidyl-tRNA. Release factor 1 (RF1) thereby recognizes UAG and UAA codons, whereas release factor 2 (RF2) is specific for UGA and UAA. After release of the peptide, the class II release factor RF3 binds the ribosome and dissociates RF1/2 from the ribosome.

RF1/2 share highly conserved regions and consist of four domains with domains 2,3 and 4 of the factors overlapping with the binding site of A-site tRNA.(Zhou et al., 2012a). For a long time, it remained elusive how these decoding factors are capable of discriminating between the different stop codons or if they indirectly recognize stop codons through interactions with the ribosome. Swapping of conserved domains between RF1 and RF2 revealed the presence of a crucial tripeptide motif, namely P(A/V)T in RF1 and SPF in RF2, located in a loop of domain 2 (Ito et al., 2000).

Exchanging these motifs between both RFs changes the specificity towards the stop codon suggesting that the tripeptide motif efficiently deciphers stop codons, in an anticodon-like manner (Ito et al., 2000; Nakamura et al., 2000).

Four high-resolution crystal structures of RF1/2 bound to both their respective stop codons explain the molecular mechanism behind deciphering stop codons (Ito et al., 2000; Korostelev et al., 2008, 2010; Laurberg et al., 2008). P(A/V)T/SPF motives are located in loops that are directed towards the decoding site, interacting with the stop codon. The stop codon itself adopts an unusual conformation with the first two bases stacking and the third base being sandwiched between G530 and residues of the release factors. Surprisingly, only a single amino acid in both motifs is in direct contact with the second position of the respective stop codon, namely the T186 for RF1 and the S206 for RF2 (Korostelev et al., 2008, 2010; Laurberg et al., 2008;

Weixlbaumer et al., 2008). This is in agreement with studies from Ito et al., showing a prerequisite of those two aa in overexpressed RF1/2 mutants in ∆RF1 or ∆RF2 strains (Ito et al., 2000). The acceptance of an A and a G for RF2 might be due to the potential of serine to interact with A and G at this position.

32 U1 position of the stop codon is recognized by backbone elements of the decoding factors that interact with N3 of uridine and explains the restriction to U at this position (Korostelev et al., 2008, 2010; Laurberg et al., 2008; Weixlbaumer et al., 2008). Due to the backbone interaction, mutations failed to confirm this interaction.

However, by introducing non-canonical RNA residues at the first position, Erlacher and coworkers were able to show that this interactions relies on the exocyclic group of uridine, explaining its exclusiveness at this position (Hoernes et al., 2018). RF1 monitors the third position via interactions of Thr194 and Q181, whereas RF2 interactions depends on T194 (Korostelev et al., 2008, 2010; Laurberg et al., 2008;

Weixlbaumer et al., 2008). Mutational studies show that exchange of aa adjacent to the tripeptides motif can change the specificity of RF1/2 (Ito et al., 1998; Korkmaz and Sanyal, 2017; Young et al., 2010). These findings, however, should not question the importance of the tripeptide motives, but rather highlight an elaborate network of interactions in which the P(A/V)T/SPF motif is a prerequisite (Figure 7A+B).

Using metal ion fluorescence resonance energy transfer, Trappl et al could show that upon recognition of the stop codon, RF1 opens from a closed to an open extended conformation on the ribosome. In contrary, this induced fit does not happen in the presence of a sense codon (Trappl and Joseph, 2016). Structures of the isolated decoding factors reveal a tight packing of domain 2 and 3 against each other, whereas bound to the ribosome, domain 3 escapes this packing and is orientated towards the PTC (Korostelev et al., 2008, 2010; Laurberg et al., 2008; Shin et al., 2004;

Vestergaard et al., 2001; Weixlbaumer et al., 2008). Opening of domain 3 requires a rearranged state of a switch loop within RF1/2, connecting domain 3 and 4. This rearranged state of the switch loop is stabilized by residues of the decoding site that adopt an alternative conformation upon stop codon recognition by release factors (Figure 7C) (Korostelev et al., 2010; Laurberg et al., 2008). Thus, recognition of the stop codon is coupled to the opening of release factors on the ribosome (Figure 7D).

Sequence alignments between all kingdoms showed the abundance of a GGQ motif in all release factors (Frolova et al., 1999). In bacteria this motif is found within the tip of domain 3, placing it next to A76 of the peptidyl-tRNA upon release factor opening. Mutations affecting the first and second glycine abolished hydrolysis, whereas mutations of glutamine were tolerated, indicating a direct involvement of the motif for hydrolysis (Frolova et al., 1999; Shaw and Green, 2007). This came as a surprise as the glutamine is post-translationally methylated in vivo leading to enhanced

33 activity for peptide release (Dincbas-Renqvist et al., 2000; Heurgué-Hamard et al., 2002). From structural analysis it came apparent that the side chain of Gln of the GGQ motif in RF1 is orientated away from A76 and the catalytic center. The backbone NH, however, is in hydrogen bonding distance to the 3’OH of A76. Mutation of Gln to Pro in RF1 eliminates the NH backbone interaction and thereby abolishes the activity of RF1 (Figure 7E) (Santos et al., 2013; Shaw and Green, 2007). Recent structures, that used release factors carrying the methylated glutamine, showed a tighter packing of the glutamine against its neighboring residues and thereby additionally stabilizing the backbone of the glutamine (Pierson et al., 2016; Zeng and Jin, 2016). The two glycines, on the other hand, are not directly involved in catalysis but are important for the conformation and integrity of the loop (Laurberg et al., 2008; Shaw and Green, 2007).

Figure 7. Termination of translation in the presence of a stop codon. (A) Decoding of the stop codon (green) by the P(A/V)T motif of RF1 (yellow). (B) Same as (A) but in the presence of RF2 and the corresponding SPF motif. (C) Comparison of the switch loop region of RF2 (red) in its open, ribosome bound conformation (yellow) and closed conformation (pink) as observed in solution. Remodeling of the switch loop on the ribosome involves stacking interaction between A1492 (16S, blue) and the switch loop.

(D) After remodeling of the switch loop of RF2 (yellow) the tip of domain III gets positioned into the PTC. This involves a movement of 60 Å compared to the closed RF2 conformation (pink). (E) Backbone interaction between the catalytic important Q230 of RF2 (yellow) and A76 of P-tRNA (orange) is shown. (F) Crystal structure of RF3 (orange) bound to a rotated ribosome. Pictures adapted from (Laurberg et al., 2008; Zhou et al., 2012b)

34 Likewise, during peptide bond formation, the presence of an A-site substrate induces a similar conformational change within the PTC (Shaw and Green, 2007). In the presence of RF1/2 this includes residues U2506, which overlaps with the binding site of RFs and U2585 which moves away from the ester bond allowing its hydrolysis by a water molecule (Shaw and Green, 2007; Schmeing et al., 2005b). This leads to the following situation: Accommodation of domain 3 and conformational changes within the PTC allow the activation of an attacking water molecule. The backbone of Gln interacts via its NH-group with the 3’OH of A76, whereas its side chain is shielding the PTC from other nucleophiles larger than water (Figure 7E) (Jin et al., 2010; Korostelev et al., 2008, 2010; Laurberg et al., 2008; Shaw and Green, 2007; Shaw et al., 2012;

Weixlbaumer et al., 2008). Similar to peptide bond formation, this reactions proceeds through a tetrahedral intermediate (Korostelev et al., 2008, 2010; Laurberg et al., 2008;

Trobro and Åqvist, 2009; Weixlbaumer et al., 2008). Break down of this state results in deacylated tRNA and free peptide. In contrast to peptide bond formation, hydrolysis of the peptide is less understood. One model suggests that 2’OH of A76 acts as a protein shuttle by accepting a proton from the nucleophilic water and subsequently transferring it to the leaving group on the 3’OH, that is stabilized by the main chain amide of the glutamine (Schmeing et al., 2005a; Shaw et al., 2012; Sievers et al., 2004; Trobro and Åqvist, 2009; Weinger et al., 2004). Other models suggest a step-wise proton transfer with only one proton moving at the same time (Kuhlenkoetter et al., 2011).

After hydrolysis the class II release factor RF3 binds the ribosome and stimulates the release of RF1/2 (Freistroffer et al., 1997; Goldstein and Caskey, 1970).

Like EF-Tu and EF-G, RF3 is a translational GTPase (traGTPase) and binds the ribosome preferentially in complex with GTP (Figure 7F) (Adio et al., 2018; Koutmou et al., 2014a; Peske et al., 2014). Additionally, the binding site of RF3 overlaps with their position on the ribosome (Gao et al., 2007; Jin et al., 2011; Pallesen et al., 2013;

Zhou et al., 2012a, 2012c). Hence, a direct interaction between RF3 and RF1/2 is unlikely as there is no overlap in the binding site, suggesting that RF3 promotes dissociation of RF1/2 indirectly (Gao et al., 2007; Jin et al., 2011; Pallesen et al., 2013;

Zhou et al., 2012a, 2012c). From crystal structures it is evident that in the absence of RF3, but presence of RF1/2, the ribosome adopts a non-rotated state (Jin et al., 2010;

Korostelev et al., 2008; Laurberg et al., 2008; Weixlbaumer et al., 2008). By contrast, it could be shown that in the presence of RF3, but absence of RF1/2, the ribosome is in a rotated state (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012c). Hence, it is

35 likely that RF3 dissociates RF1/2 by inducing subunit rotation. Recent biophysical studies further refined the understanding for this process. Like EF-Tu, RF3 binds the ribosome preferentially in complex with GTP (Adio et al., 2018; Koutmou et al., 2014b;

Peske et al., 2014). Binding of RF3 facilitates the transition from non-rotated RF1/2-bound state to a rotated state and thereby removing the decoding factors (Adio et al., 2018; Ermolenko et al., 2007b; Sternberg et al., 2009). The conversion from a non-rotated to a non-rotated state seems to be dependent on the presence of GTP bound to RF3, as non-hydrolysable analogues showed a reduced rate in conversion (Adio et al., 2018; Shi and Joseph, 2016). Interestingly, RF3 mutants that are able to bind GTP but are deficient in hydrolysis are also able to induce this transformation, suggesting that the presence of the hydrolysable analogue is needed but not its hydrolysis (Adio et al., 2018; Shi and Joseph, 2016). It rather seems that hydrolysis is needed to reset ribosomes back to a non-rotated state by dissociating RF3 from the ribosome (Adio et al., 2018; Peske et al., 2014; Shi and Joseph, 2016).

Last but not least it should be noted that cells lacking RF3, show no growth defect (Grentzmann et al., 1994; O’Connor, 2015). Even more, RF3 is only found in a subset of bacteria suggesting that RF3 is not part of a conserved mechanism but an auxiliary factor fine tuning the RNA machinery (Margus et al., 2007).

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