The elongation cycle requires coordination of an ordered series of events. The elongation factor 1·aa-tRNA·GTP ternary complex must first interact with the ribosomes’ factor-binding site. At the decoding center in the small subunit, the presence of cognate codon:anticodon base-pairing is transduced up the body of the aa-tRNA, activating GTP hydrolysis by elongation factor 1 (Valle et al., 2002
; Cochella and Green, 2005
). This initiates accommodation of the aa-tRNA into the PTC. Successful accommodation has been hypothesized to be accompanied by rRNA structural changes that both close the aa-tRNA accommodation corridor (Sanbonmatsu et al., 2005
), and activate the PTC (Schmeing et al., 2005
). Concurrently, the structure of the factor binding site must also change to favor elongation factor 2 binding, setting the stage for translocation (Sergiev et al., 2005
). The rRNA structure probing and ribosome binding data presented in the current study suggest a model describing how L3 may help to coordinate these ribosome-associated functions by acting as a “gatekeeper” to the A-site.
Computer modeling studies suggest that during accommodation, the aa-tRNA 3′ end moves through a ‘corridor’ formed by helix 89 on one side and the helix 90 – 92 structure on the other (Sanbonmatsu et al., 2005
). The chemical protection data clearly shows that specific bases in the helix 90 – 92 side of this pathway are generally deprotected in the W255C mutant as compared to wild-type ribosomes. Thus, the increased accessibility of bases in this structure to small chemicals suggests that it is in a relatively ‘open’ conformation (). The computational simulations of aa-tRNA accommodation also suggest that the aa-tRNA 3′ end passes through two rRNA-based structures or “gates”. The first gate encountered by aa-tRNA (gate 1) is formed by U2491 and C2556 (yeast U2860 and C2924), while the second (gate 2) is formed by C2573 (yeast C2941) (see ). Notably, the gate bases in the helix 90 – 92 structure (C2924 and C2941) were deprotected, consistent with an open conformation. Interestingly, U2861 was protected from chemical attack. It is possible that reorientation of the helix 89 gate 1 base U2860 had a shielding effect on U2861, consistent with the idea that the W255C mutant promotes opening of the accommodation corridor. Additionally, the chemical protection studies suggest that these conformational changes in this, and other mutants, are promulgated along the entire pathway traveled by the aa-tRNA 3′ end, from the SRL down to the PTC ().
Opening of the aa-tRNA accommodation corridor should affect the biochemical properties of the ribosome. Specifically, such changes should promote increased affinity for aa-tRNA, which is supported by the observation of decreased Kd values for aa-tRNA by the W255C, R, K, and H256Q mutants. The increased eEF2 Kd values for these mutants are also consistent with the notion that ternary complex and eEF2 binding should be mutually exclusive. This is further supported by the H256A and H259A mutants, which promoted increased affinity for eEF2 at the expense of their ability to interact with aa-tRNA. These biochemical observations suggest that two classes of mutants were generated: one promoting ribosomal conformations more favorable to eEF2 binding, the other favoring binding of the eEF-1A·aa-tRNA·GTP ternary complex. These data suggest that in the L3 mutants, bases in the PTC and along the aa-tRNA accommodation pathway have been artificially shifted into conformations that are normally transitory during translation in wild type cells. Specifically, we propose that some W-finger mutants (e.g. W255C, R, K, and perhaps H256Q) favor positioning of the SRL into the eEF-1A·aa-tRNA·GTP binding state, and the accommodation corridor into the “open” conformation, while others (H256A, H269A) favor the closure of the corridor and eEF2 binding.
These findings suggest a model wherein the W-finger may normally assume different positions within the ribosome during these two factor-binding states, possibly accompanied by different patterns of putative interactions between amino acids in the W-finger and 25S rRNA bases. This is cartooned in . By this model, movement of the L3 W-finger may participate, either actively or passively, in an allosteric process to coordinate these events. Specifically, positioning of the W-finger toward its N-terminal side, i.e. directed away from the PTC and toward the factor binding site, may help to both open the helix 90 – 92 face of the accommodation corridor, and to position the SRL so as to favor binding of the eEF-1A·aa-tRNA·GTP ternary complex. We suggest that while in this conformation, A2940 of 25S rRNA may interact with H259 of L3. The model proposes that during accommodation, the W-finger swings toward its C-terminal side (toward the PTC) and that the A2940-W255 interaction is established upon completion of aa-tRNA accommodation into the A-site. Additionally, since the aa-tRNA and eEF2 Kd values for the H256A mutant were between those observed for the W255 mutants (W255C, R, and K) and H259A, it is also possible that interaction between A2940 and H256 could constitute a transitional intermediate between the two states. We propose that during rearrangement of the W-finger, the helix 90 – 92 structure moves to close the accommodation gates. Concurrently, the SRL atop helix 95 is repositioned so as to form the eEF2 competent binding site. This sequence of events would allow the occupancy status of the A-site to be communicated to the translation factor binding region, and prevent entry of new aa-tRNA into the A-site during translocation. It must be noted that our results have only revealed amino acid residues in the W-finger that may be involved in these conformational events, not their rRNA counterpart bases. Although the chemical properties of the substituted amino acids suggest their involvement in interactions with nucleotides, the actual rRNA base involved was not experimentally determined, but A2940 represents the most likely candidate based on the crystal structures.
Model for the role of L3 in coordinating functions of the elongation factor binding site: L3 as the gatekeeper to the A-site
Resistance of the W255C mutant to anisomycin has been known for a many years (Fried and Warner, 1981
), but the underlying mechanism has been unclear. Early studies showed that the W255C mutation did not affect anisomycin binding (Jimenez and Vazquez, 1975
), suggesting that structural and/or conformational changes in resistant ribosomes may reduce the ability of anisomycin to compete with aa-tRNA for entry into the A-site. The chemical protection data presented in the current study show structural changes in the large subunit consistent with a more “open” conformation in the vicinity of the A-site of the PTC and in the aa-tRNA accommodation corridor in the anisomycin resistant mutants. Opening of the accommodation corridor should facilitate aa-tRNA passage into the A-site, consistent with the decreased Kd
values for aa-tRNAs. In contrast, because of its small molecular radius relative to aa-tRNA, access to the PTC by anisomycin should not be as intrinsically limited by the two gates, and hence should not be stimulated as much by gate opening. Therefore, it is possible that these mutations lower the barriers encountered by aa-tRNA relative to anisomycin during accommodation, thus altering the kinetic partitioning ratios for these two molecules, resulting in anisomycin resistance. An alternate, non-exclusive explanation may be that changes in the interactions between anisomycin and bases in the PTC (see ) consequent to these mutations may contribute to anisomycin resistance. Interestingly, the –OH group of the N-terminal serine of L3 (S2) is predicted to interact with C2924, the helix 90 ”Gate 1” residue (see ). The S2T mutant was also strongly anisomycin resistant (Meskauskas et al., 2005
), and experiments are ongoing characterizing a bank of S2 mutants.
An “entropic catalysis” model of peptidyltransfer posits that precise positioning of substrates and water reorganization in the PTC allows formation of a highly ordered electrostatic network that stabilizes reaction intermediates, providing the proper environment for the ribosome to accelerate peptide bond formation (reviewed in Rodnina et al., 2007
). The rRNA structure probing experiments in the current study are consistent with this model insofar as distortion of the topology of the A-site side of the PTC by the W-finger mutants could affect positioning of the aa-tRNA 3′ end, promoting the observed peptidyltransfer defects. It has also been proposed that the PTC is activated by an ‘induced fit’ mechanism, wherein accommodation of the aa-tRNA acceptor stem into the A-site reorganizes the geometry of the PTC, making the ester group of the peptidyl-tRNA accessible for nucleophilic attack (Schmeing et al., 2005
). The involvement of the L3 W-finger in this process remains an intriguing possibility.
It is remarkable how atomic scale allosteric events can propagate themselves all the way to the biological level. Translating our understanding of molecular structure and biochemical properties of molecules may lead to concrete medical benefits. For example, understanding how these mutants affect the interactions between ribosomes and trans
acting factors, and how this leads to resistance to A-site specific antibiotics can provide the foundation for the design of a new antibacterials to counter resistance of pathogenic bacteria to drugs such as tiamulin (Pringle et al., 2004
). This research may also aid in the development of new antivirals insofar as it furthers our understanding of how changes at the atomic level can affect peptidyltransfer, and hence –1 PRF and virus propagation. It is hoped that atomic resolution structures of yeast ribosomes will be solved in the near future, and that achievement of this milestone will enable the models proposed herein to be directly visualized.