The Sarcin–Ricin Loop

The sarcin–ricin loop is one of the most conserved rRNA regions of the ribosome, which underlines its importance in ribosome function. It is located in helix 95, in domain VI of 23S/25S/28S rRNA (nucleotides 2646-2674 in *E. coli*, 3012-3042 in yeast) (Figure 2A).

**Figure 2.** The model of sarcin–ricin loop structure. (**A**) Alignment of highly conserved secondary structures of yeas<sup>t</sup> and *E. coli* SRL. The red color indicates the key adenine hydrolyzed by the ricin. A conserved fragment of 12 nucleotides is marked with a gray color. (**B**) Structure of the *S. cerevisiae* SRL (PDB code 3U5H). The key adenine was marked in red (A3027). The individual structural elements of the SRL - the stem, the flexible region, the G-bulged cross-strand stack with the highlighted individual nucleotides and the GAGA loop - are marked with a dotted black line. The gray fields show the non-canonical π-stacking interactions between particular bases. Model prepared by PyMol software, (The PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC, NY, USA) based on [42].

Unlike other rRNA regions, which form compact structures coordinated by rRNA–rRNA or rRNA-protein interactions, the SRL exists on the ribosome as an autonomous unit [43–45] and is exposed to the solvent [46]. This unique feature is critical for its accessibility for external factors like trGTPases. From the structural point of view, the SRL has the conformation of a distorted hairpin [47]. It consists of several well-organized elements (Figure 2B) [48]: the stem, the flexible region, the G-bulged cross-strand stack, and the GAGA loop [45] with the key adenine base (A2660/A3027—*E. coli*/*S.cerevisiae* numbering), which is a target for ricin activity. The SRL stem structure is formed mainly by classical Watson–Crick base pairs, whereas the rest of the structure is stabilized mainly by π-stacking interactions. The critical element, the GAGA tetra-loop, forms a compact well-organized structure [43]. The spatial organization of the loop structure is determined by non-canonical interactions between base pairs, allowing the carbohydrate-phosphate backbone of RNA to form the atypical spatial form, which is recognized by translational factors [43]. The critical bases A2660, G2661, and A2662 in the GAGA loop (*E. coli* numbering) associate with each other via non-canonical π-stacking interactions that stabilize the loop structure [42,43]. The crucial A2660, located at the top of the hairpin structure, is stabilized via the stacking interactions with G2661 (Figure 2B). Importantly, A2660 is fully exposed and does not form any hydrogen bonds with other bases of the loop, making it easily accessible to external factors like ricin [49]. Biochemical studies on prokaryotic and eukaryotic ribosomes have shown that the SRL represents a critical element responsible for the interaction and stimulation of all trGTPases activity [50,51]. Additionally, it has been early recognized that this structure represents the main target for numerous toxins [50,52–54]. Recent structural analyses have brought detailed insight into the intricate interplay between the SRL and trGTPases and at the same time cast light on the molecular aspects of ricin toxicity [28,29,40,55]. In general, all trGTPases interact with the SRL via the GTP-binding domain (G-domain) comprising the active site of the factor, responsible for GTP hydrolysis [17]. It should be stressed that the structure of the G-domain is evolutionarily conserved among all trGTPases, and biochemical and structural investigations support the notion that the mechanism of activation of GTP hydrolysis by the ribosome is universally conserved [17,56]. The trGTPases convert chemical energy into mechanical forces at the expense of GTP hydrolysis, and this drives the ribosome through the translational cycle. The key role in catalysis is played by an invariant histidine residue (His84 in EF-Tu, His87 in EF-G, or His61 in SelB, or His108 in eEF2) [56–60]. This histidine may adopt two conformations: an inactive "flipped-out" state (pointing away from the γ-phosphate of GTP) and an active flipped-in state (reaching towards the γ -phosphate) (Figure 3). After joining the factor to the ribosome, the SRL is "inserted" into the catalytic center of the G-domain, and the phosphate moiety of A2662 coordinates, by means of electrostatic interactions, the catalytic histidine positioning to the active "flipped-in" position towards the γ-phosphate of GTP [28,38,61]. The positively charged histidine points towards the water molecule aimed at the nucleophilic attack on GTP γ-phosphate [29,38,58,62]. Additionally, the phosphate of A2662 coordinates a Mg<sup>2</sup>+ ion, important in positioning of the Asp residue (Asp21 in EF-Tu, Asp22 in EF-G, Asp10 in SelB), which is also crucial for GTP hydrolysis (Figure 3) [28,58].

**Figure 3.** Model of GTP hydrolysis activation with the aid of sarcin–ricin loop (SRL), with the EF-G as trGTPase. Left panel: the organization of the active site in isolated EF-G. Asp22 and His87 are in "flipped-out" state, pointing away from GTP; the "hydrophobic gate" formed by amino acids Ile63 and Ile21 prevents His87 from adopting the active conformation. Right panel: the reorganization of the active site of EF-G as a result of binding of EF-G to ribosome and inserting the SRL to the G-domain. Base A2660 interacts with His20, which induces Ile21 movement away from GTP and "hydrophobic gate" opening. The phosphate of A2662 directs His87 and Asp22 residues (through Mg<sup>2</sup>+) to "flipped-in" conformation which allows for water molecule activation and GTP hydrolysis (see the details in the text).

Recently, A2662 and G2661 within the tetraloop structure were distinguished as critical elements, directly involved in stimulation of the GTP hydrolysis process [28,38]. Interestingly, α-sarcin cleaves the bond between eukaryotic nucleotide equivalents to *E. coli* G2661 and A2662, which results in ribosome inactivation [63,64]. The A2660 base, which is cleaved-off by ricin, plays a distinct but weighty function, which could be named as the "power behind the throne" title role. As shown based on the structure of the EF-G–ribosome complex in a pre-translocation state, an intricate network of hydrogen-based interactions involving the G2661 and A2660 of the SRL, EF-G (Glu456, Arg660, Ser661 and Gln664), and ribosomal L6 (Lys175) is formed in the immediate vicinity of the GTPase active site, with A2660 being the central element of the network [40]. Thus, depurination of A2660 may prevent the surrounding elements from adopting the active conformation, which is required to bind the metal ions necessary to stabilize Asp22 and neighboring regions of EF-G in the activated form [55,65]. On the other hand, A2660, together with G2661, plays a crucial role in opening of a so-called hydrophobic gate, which prevents the invariant His residue in the free trGTPase from achieving an active state and spontaneous GTP hydrolysis. In the complex of the EF-G/ribosome, the bases A2660 and G2661 interact with His20 of EF-G, which in turn interacts with Ile21, forming a hydrophobic gate with Ile63. These interactions contribute to its opening and repositioning the His87 into its active position [40]. The structural analyses of the A2660 role are supported by biochemical insight. It has been shown that lack of the single exocyclic N6 amino group at position 2660 within rRNA inhibited GTP hydrolysis on the EF-G/ribosome complex. Importantly, the introduction of different exocyclic groups with dissimilar chemical groups, such as inosine, dimethyladenosine, or even 6-methylpurine, restored

the GTP hydrolysis activity of EF-G. The experimental biochemical data indicate that the critical favorable chemical feature is related to electron configuration that allows participation in the aromatic π-electron interaction system of the purine, which in turn facilitates the π-stacking e ffect [66]. It should be underlined that despite the vast number of data collected from only the bacterial model, the amino acid sequence, called PGH motif (with the invariant His residue) is universally conserved and present in EF-Tu, EF-G, IF2, and RF3, as well as archeal and eukaryotic trGTPases [67]. What is more, the highly conserved A2660 base moiety (A3027 - yeast, A4324 - rat, A4605 - human) within the tetra-loop of SRL [56] represents the most crucial base which contributes to a cooperative interaction network, which stabilizes the active state of trGTPases, promoting GTP hydrolysis.
