Another Look at Mutations in Ribosomal Protein S4 Lends Strong Support to the Domain Closure Model

Abstract

Ribosomes employ a "kinetic discrimination" mechanism, in which correct substrates are incorporated more rapidly than incorrect ones. The structural basis of this mechanism may involve 30S domain closure, a global conformational change that coincides with codon recognition. In a direct screen for fidelity-altering mutations, Agarwal Ribosomes synthesize proteins on the basis of mRNA templates by using a diverse pool of aminoacyl-tRNA (aa-tRNA) substrates. In each round of elongation, EF-Tu catalyzes the binding of aa-tRNA to the A site of the translating ribosome (carrying P-site peptidyl-tRNA) in a process termed decoding. Once aatRNA moves completely into the A site, peptidyl transfer occurs, lengthening the nascent chain by one residue. EF-G then catalyzes translocation, the movement of the tRNAs (and paired codons) to their adjacent ribosomal sites. This leaves the A site vacant, ready for the next decoding event.During decoding, aa-tRNA binds the ribosome as part of a ternary complex with EF-Tu and GTP (reviewed in reference 1). Initial binding of EF-Tu-GTP-aa-tRNA mainly involves contacts with the 50S subunit and is followed by a sampling of codonanticodon interactions on the 30S subunit. Codon-anticodon pairing in the 30S A site leads to GTPase activation and GTP hydrolysis, allowing release of aa-tRNA from EF-Tu. The aa-tRNA then either moves completely into the A site (a step termed accommodation) and participates in peptide bond formation or dissociates from the ribosome.The ribosome selects cognate aa-tRNA from all other aatRNAs with high speed (Ͼ20 s Ϫ1 ) and fidelity (error rate, ϳ10 Ϫ4 ) (2). The high fidelity is explained in part by a kinetic proofreading mechanism whereby differences in substrate binding affinity are exploited twice to increase the overall level of discrimination (3-5). In essence, the functionally irreversible GTP hydrolysis step of the pathway provides a second opportunity for rejection of nearcognate aa-tRNA. This proofreading mechanism, though, is not maximally exploited for fidelity (1, 6). Instead, the ribosome additionally employs a "kinetic discrimination" mechanism to achieve both speed and fidelity. Cognate codon recognition accelerates GTPase activation/GTP hydrolysis and accommodation (7-10). This allows rapid incorporation of cognate aa-tRNA specifically, obviating the need for substrate binding equilibria to be approached.An important question is how codon recognition stimulates GTPase activation. Cognate codon-anticodon pairing results in rearrangement of rRNA nucleotides G530, A1492, and A1493, which dock into the minor groove of the codon-anticodon helix (11,12). These changes in the 30S A site are somehow transmitted 80 Å to the GTPase domain of EF-Tu. One potential conduit for signaling is the tRNA itself, which is known to adopt a distorted conformation in the GTPase-activated state (13-15). Another non-mutually exclusive possibility is that signaling occurs through the 30S subunit. Crystallographic studies of the 30S subunit suggest that cognate A codon re...

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

“…A recent reevaluation of S4/S5 mutations showed that all C-terminal truncations of S4 confer a ram phenotype (Agarwal et al 2015). These truncations undoubtedly destabilize the open conformation of the subunit, lending strong support to the idea that domain closure contributes to GTPase activation (Ogle and Ramakrishnan 2005;Voorhees et al 2010;Fredrick 2015). Ram mutations in h8/h14 act by disrupting bridge B8.…”

“…Genetic and structural evidence suggest that formation of the closed (on) state involves not only rearrangement of the A site but also conformational changes elsewhere, such as disruption of bridge B8 and inward rotation of the 30S shoulder (Ogle and Ramakrishnan 2005;McClory et al 2010;Fagan et al 2013;Fredrick 2015). Binding of tRNA to the 30S A site promotes the open-to-closed transition, and vice versa, as depicted in the thermodynamic cycles of Figure 4.…”

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

“…By establishing that the ΔV200 and E201* uS4 alterations in both E.coli and S. enterica have a ram phenotype, the results presented here help resolve a puzzling inconsistency in the literature [8]. With the exception of ΔV200 and E201 and Q53L in S. enterica all other uS4 mutants isolated as suppressors of the streptomycin dependence, or the slower growth phenotypes associated with uS12-alterations have proved to be error-prone.…”

“…However, at least some interface-disrupting uS4 mutants have an error-restrictive phenotype, which is difficult to reconcile with the open-closed model [8]. These uS4 mutations, isolated in Salmonella enterica carry a deletion of Val200 (ΔV200) or a truncation at Glu201 (E201*) [9].…”

Intersubunit Bridges of the Bacterial Ribosome

“…For example, codon-anticodon pairing in the 30S A site induces movement of the 30S head and shoulder domain towards each other around the A site, resulting in a “closed” conformation 9 , which is thought to activate elongation factor EF-Tu during the decoding process 10; 11; 12; 13 . Moreover, two major large-scale rearrangements, rotation of 30S head domain relative to the rest of the subunit, as well as the rotation between 30S and 50S subunits, have been attributed to various steps of translation, including subunit joining 14 , translocation 15; 16; 17; 18; 19; 20; 21 , peptide release 22; 23 , and ribosome recycling 24 .…”