Mutational Analysis of S12 Protein and Implications for the Accuracy of Decoding by the Ribosome

Abstract

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

“…It should be noted that, unlike the better characterized rpsL variants (Zaher and Green 2010), the A1913U ribosomes exhibit no apparent defects during cognate tRNA incorporation. We argue that a restrictive phenotype is the likely outcome of site-directed mutations targeting key contact points for tRNA selection, since certain steps of closure that are critical to forward steps in the process are likely made less efficient (Sharma et al 2007). Our in vitro selection approach allowed us to identify tRNA Trp variants that more potently miscode and would thus be classified as having a ribosomal ambiguity (or ram) phenotype.…”

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

“…Here we describe streptomycin-resistance mutations in ribosomal protein S12 at sites distant from the streptomycinbinding site and positions of other previously identified resistance mutations (Carter et al 2000;Gregory et al 2001;Sharma et al 2007). One of these substitutions, H76R, is located in a QEH triplet consisting of residues Q74, E75, and H76, which has been interpreted in low-resolution cryo-EM maps as a protrusion contacting the aminoacyltRNA acceptor helix at or near residue U69 (Stark et al 2002;Valle et al 2003).…”

Antimycobacterial Activity Some Different Lamiaceae Plant Extracts Containing Flavonoids and Other Phenolic Compounds

“…STR is a protein synthesis inhibitor. STR interacts with a 30S subunit of ribosome and disrupts protein synthesis (Sharma et al, 2007;Springer et al, 2001). Its mechanism of action starts with binding tightly to the phosphate backbone of 16S rRNA in different domains and making contact with the S12 ribosomal protein; finally it causes misreading of the bacterial genetic code during translation (Carter et al, 2000;Hosaka et al, 2006).…”