With atomic resolution ribosomes in hand, the major challenge is linking structure to function. Three significant questions in the field are: ‘how does the ribosome coordinate the sequential processes of translation elongation’; ‘what are the allosteric signals that activate the peptidyltransferase center’; and ‘what are the functions of the extended domains of ribosomal proteins?’ Recent work from our laboratory demonstrated that the central extended domain, or ‘W-finger’ of L3 acts as a molecular switch to help coordinate binding of the two elongation factors, leading us to describe L3 as the ‘gatekeeper’ to the ribosomal A site (20
). The focus of the current study is the second extended domain located at the N-terminus of L3. Here, a combination of molecular genetics, biochemistry, rRNA structure probing and molecular modeling approaches were used to address these questions.
As noted above, the N-terminus of L3 appears to form a hydrogen bond with C2924 (E. coli
C2556), which in turn is one of the two partners of the first accommodation gate. We suggest the N-terminal extension of L3 may act as a piston to either directly induce or indirectly support conformational changes in this accommodation gate. We further suggest that this interaction is broken in the S2 series of mutants, interfering with its ability to fully open or close, while lengthening it by insertion of an additional amino acid forces the accommodation corridor into more closed conformation. The changes in the vicinity of the other side of the first gate [around U2860 (E. coli
U2492)] and at the second gate [in the vicinity of C2941 (E. coli
C2573)] also suggest that the coordination between the two gates is inhibited by these mutants. Importantly, one of these bases, A2940 (E. coli
A2572), appears to interact with the W-finger of L3. The deprotection of C2924 (E. coli
C2556), A2940 and C2941 by the W255C mutation (20
) suggests that both the N-terminal and central extensions of L3 work in concert to coordinate opening and closing of the aa-tRNA accommodation corridor.
Together with the studies on the W-finger domain, we propose a more detailed, mechanical model of L3 functioning as a ‘rocker switch’ to help coordinate an allosteric signaling pathway between the elongation factor binding site and the peptidyltransferase center. This model is cartooned in . Starting with the open conformation (, left side), positioning of the N-terminal extension away from the accommodation corridor pulls C2924 (E. coli
C2556, the Helix 92 gate 1 base) along with it, away from U2861 (E. coli
U2493, the Helix 89 gate 1 base). This favors closure of the proximal loop in Helix 89 (protecting these bases), and pulls bases along the H90–92 structure away from the corridor, exposing them to solvent (deprotection). The fully open conformation positions Helices 89, 90–92, and 95 to form the binding site for the aa-tRNA•eEF1A•GTP ternary complex, promoting increased affinity for aa-tRNA. We suggest that the ‘more open than closed’ conformation of the S2T and S2K mutants accounts for their increased affinities for aa-tRNA and anisomycin resistance. Notably, the fewer number of changes in rRNA structure promoted by the S2A mutant suggests a reason for its lack of effect on aa-tRNA binding and anisomycin sensitivity. In the fully open conformation, the W-finger is in the ‘extended’ conformation, where its tip occupies the A site of the PTC. In this conformation, H256/H259 interact with A2940 of 25S rRNA (E. coli
A2572, H. marismortui
U2607). This is mimicked by mutations to W255, which also increase ribosomal affinity for aa-tRNA presumably by inducing the open conformation of accommodation corridor (20
Figure 5. L3 functions as a ‘rocker switch’ to coordinate elongation factor binding, aa-tRNA accommodation and PTC activation. (Left) Ribosome in ground state with P site occupied by peptidyl-tRNA. L3 W-finger is in the ‘extended’ (more ...)
Accommodation of aa-tRNA into the A site displaces the W-finger, repositioning W255 to interact with A2940 (E. coli
A2572), a state which is favored by mutagenesis of either H256 or H259 to alanine (20
). We propose that this movement is transduced through the globular domain of L3, which in turn pushes the N-terminal extension toward the accommodation corridor. This in turn causes the H90–92 structure to move to close the accommodation corridor (with the resulting changes in protection patterns along this structure) and away from the SRL (resulting in its deprotection). We suggest that this state is somewhat mimicked by the iG12 mutant, although as noted above, this mutant is not able to fully close due to its having broken the coordination between the two gates. The closed state provides the structural basis for eEF2 binding (perhaps by making the SRL available for binding) as evidenced by the increased affinity of iG12 (this study), H259A and H256A (20
) mutants for this elongation factor. In addition, closing of the accommodation corridor conferred protection from chemical attack on the bases in Helix 89 that interact with the N-terminal ‘hook’ extension of L10. In light of the data presented in the accompanying manuscript (see accompanying paper by Petrov et al.
), we suggest that this provides a point of information transfer between L3 and L10 in helping to coordinate the different functions performed by the large subunit.
The reciprocal relationship between aa-tRNA and eEF2 binding appeared to be partially decoupled by the S2T and S2K mutants. We suggest that the explanation for this lies in the decreased strength of the interaction between the N-terminal extension and C2924 (E. coli
C2556). We hypothesize that the resulting ‘more open than closed’ conformation allows increased affinity for aa-tRNA, but does not stabilize interaction between the loop of H91 and the SRL to the extent that significant inhibition of eEF2 binding was observed. However, this conformation of the accommodation corridor should enhance entry of aa-tRNA into the A site, consistent with the decreased KD
values for the S2T and S2K mutants for aa-tRNA. As noted previously (20
), the ability of anisomycin to access the A site should not be as sensitive to the conformational status of the accommodation due to its much smaller molecular radius. Thus, in these mutants, the kinetic partitioning ratio between anisomycin and aa-tRNA is lowered, providing an explanation for their drug resistance.
Examination of Supplementary Figures 1 and 2
reveals that only the eukaryotic and archael L3 proteins contain the N-terminal extension, and that no comparable structure exists in bacteria. Why might this be? Although the nuclear location signal is situated in this extension in eukaryotes, the absence of a nucleus in the Archaea
suggests that the N-terminal extension originated for another reason. One possible explanation may come from the observation that N-terminus of bacterial ribosomal protein L27 extends into the PTC, where it is predicted to interact with the A76 phosphate of the A-site tRNA (44
). Deletion of the N-terminus of L27 resulted in reduced rates of peptidyltransfer and decreased affinity for aa-tRNA (52
). Eukaryotes and Archaea
do not have a homologous protein. Thus, it is possible that they evolved the N-terminal extension of L3 to fulfill a similar function. A second explanation is suggested by the fact that the therapeutic basis for many antibiotics derives from their specific inhibition of bacterial as opposed to eukaryotic ribosomes (53
). Examples specific to the large subunit include chloramphenicol, many of the macrolides and the streptogramin group of antibiotics (54
). Thus, it is possible that the N-terminal extension of L3 may affect the structure of the A-site to favor aa-tRNA binding over that of antibiotics, and/or better coordinate peptidyltransfer, thus providing Archaea
and eukaryotes with a selective advantage against small molecule inhibitors of protein synthesis. Experimentally, if addition of an N-terminal extension of L3 could be tolerated by E. coli
, it would be interesting to note whether this would confer resistance to a class(s) of large-subunit-specific antibiotics. Alternatively, this could also be computationally modeled and subjected to molecular dynamics free energy simulations.
The effects of the iG12 mutant on G2921 (E. coli
G2553, H. marismortui
G2588) is of particular interest. It has been proposed that Watson–Crick pairing between this base and C75 of the aa-tRNA is a critical step in positioning of A-site substrate in the induced fit model of peptidyltransfer (55
). Consistent with this model, G2921 became relatively protected when the A-site of wild-type ribosomes was occupied by aa-tRNA ( and ). Importantly, this protection failed to occur in the iG12 mutant. This could account for the strong increase in the KD
of this mutant for aa-tRNA. Why then did the iG12 mutant have but a small effect on peptidyltransferase activity, even though its growth rate was significantly affected? We suggest that this can be explained by the fact that this parameter was monitored using puromycin instead of aa-tRNA, and that absence of a moiety like C75 on this drug served to minimize the observed effect by iG12 (we predict, that peptidyltransferase activity would be significantly reduced in this mutant if C-puromycin or aa-tRNA was used as substrate). In contrast, G2921 was strongly protected in salt-washed W255C mutant ribosomes, which did promote a very strong peptidyltransferase defect (20
). These structural data suggest that both the N-terminal extension and W-finger L3 mutants may affect the C75-G2921 interaction and thus be indirectly involved in the induced fit mechanism of peptidyltransfer (55
). Alternatively/additionally, changes in local rRNA structure affecting G2921 could cause a shift in the 3′ end of the aa-tRNA, thus misaligning it and promoting the observed defects in peptidyltransfer activity and aa-tRNA binding. Lastly, the defects of these mutants in their ability to maintain the yeast killer virus is consistent with the link between decreased peptidyltransferase activity and virus propagation (31
The pattern of viability of the S2 mutants could not be classified by any of the biophysical properties of their amino acid sidechains. Although lethality could be due to defects in the interaction between the N-terminus of L3 and C2924, the observed pattern followed the ‘N-end rule’ of post-translational modification (56
), suggesting that L3 stability may be the cause. Specifically, there was a strong correlation between amino acid identity at the second residue of the protein (Ser, Ala, Gly and Thr) and amino acids preferred for removal of the N-terminal methionine followed by acetylation in eukaryotes (49
). Curiously, this is inconsistent with mass spectroscopic analyses of yeast ribosomes, where there is strong evidence showing that while N-terminal amino acid of L3 is serine, it is not acetylated (47
) Such processed but unacetylated N-termini were also observed for many other yeast ribosomal proteins. Do these proteins simply bypass the stability requirements for co-translational N-terminal processing and acetylation, or are they initially processed and acetylated during translation, but later deacetylated for ribosome biogenesis? The latter idea is supported by the notion that ribosome assembly may be facilitated by interactions between highly basic, nonstructured extensions on ribosomal proteins and negatively charged rRNA phosphate groups. Acetylation of N-termini could interfere with assembly by reducing positive charges, potentially inhibiting RP–rRNA interactions, and promoting structural motifs in regions of RPs that need to be unstructured in order to assemble with rRNA. Thus, we suggest that although acetylation is required to stabilize RPs in the cytoplasmic compartment of the cell, this chemical modification must be removed for the process of ribosome biogenesis in the nucleolus. This in turn raises the question of the identity of the deacetylase. A prior study showed that mutation or deletion of RPD3
, better known as a histone deacetylase, and of proteins that target Rpd3p to heterochromatin but not to euchromatin, resulted in phenotypic defects similar to those observed with many L3 mutants (60
). The hypothesis that Rpd3p may also play a critical role in deacetylating ribosomal proteins prior to their incorporation into nascent ribosomes is currently being tested in our laboratory.