Distinct functional classes oframmutations in 16S rRNA

Abstract

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EFTu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.

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

“…These effects, interpreted in light of earlier work (Pape et al 1998(Pape et al , 1999Gromadski and Rodnina 2004), indicate that the mutations accelerate the GTPase activation step of initial selection (McClory et al 2010). These same mutations were also found to cause defects in the proofreading phase, reducing the rejection rate of near-cognate aa-tRNA after GTP hydrolysis typically by three-to eightfold (McClory et al 2014). The A-site mutations C1054U and C1054A were somewhat exceptional in that they altered proofreading in a tRNA-specific way.…”

“…As noted previously (McClory et al 2010), the single mutations C1200U and G1491A have a much larger effect on UGA read-through than on GAU miscoding, which cannot be readily attributed to altered RF2 activity (McClory et al 2014). These mutations in or near the A site are unique in that they increase miscoding in vitro in a context-dependent manner, causing high error rates for only particular aa-tRNAs and/or codons (McClory et al 2014).…”

“…) and thereby reduce the accuracy of aa-tRNA selection (Johansson et al 2012;McClory et al 2014;Zhang et al 2015). Ram mutations have mapped not only to the A site but also to distal regions (Fig.…”

“…Further studies of a representative subset of 16S rRNA ram mutations have shown that they confer defects in both the initial selection and proofreading phases of decoding (McClory et al 2014). Mutations in h8/h14 (h8Δ3, h14Δ2, G347U), h12 (G299A), h34 (C1054A, C1054U, C1200U), and h44 (G1491A) similarly increase k cat of GTP hydrolysis in single-turnover reactions by approximately twofold and approximately ninefold for cognate and near-cognate ternary complexes, respectively, with small (approximately twofold) increases in K M in the cognate case (McClory et al 2010(McClory et al , 2014Fagan et al 2013).…”

“…Mutations in h8/h14 (h8Δ3, h14Δ2, G347U), h12 (G299A), h34 (C1054A, C1054U, C1200U), and h44 (G1491A) similarly increase k cat of GTP hydrolysis in single-turnover reactions by approximately twofold and approximately ninefold for cognate and near-cognate ternary complexes, respectively, with small (approximately twofold) increases in K M in the cognate case (McClory et al 2010(McClory et al , 2014Fagan et al 2013). These effects, interpreted in light of earlier work (Pape et al 1998(Pape et al , 1999Gromadski and Rodnina 2004), indicate that the mutations accelerate the GTPase activation step of initial selection (McClory et al 2010).…”