Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

Abstract

Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges.

Structure of a eukaryotic cytoplasmic pre‐40S ribosomal subunit

“…The last visible residues of the linker form specific contacts with the displaced rRNA helix 31 as well as with proteins uS13 and uS19 (Figs E and EV4C). These residues, 392–408, are highly conserved in eukaryotes (Appendix Fig S3B), pointing toward a key role of this interaction with these two universally conserved ribosomal proteins, whose prokaryotic homologs are important for intersubunit bridging (Jenner et al , ; Bowen et al , ) as well as for tRNA binding (Lomakin & Steitz, ; Graifer & Karpova, ).…”

Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation

“…Interestingly, several studies in yeast identified allosteric information pathways connecting functional centers in the large ribosomal subunit (LSU) to the decoding center in the SSU through the B1a and B1b/c intersubunit bridges 21,67,68 . In eukaryotes and archaea, uS13 participates in B1b/c bridge and uS19 is part of the B1a bridge.…”

Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection

“…Instead, it is likely that the G:U wobble base pairing resulting from the C1209U mutation might have caused structural perturbation in h32 and h30a helical regions of 18S rRNA which could perturb the premature head rotation in ‘Closed/P IN ’ conformation for eIF5 G31R , eIF2β S264Y , eIF1 K60E , and eIF1A‐ΔC mutants and thereby prevent UUG start codon recognition of the HIS4 UUG transcript, thus suppressing the Sui − phenotype. Interestingly, a small ribosomal protein uS19 that interacts with helices h32 and h30a of 18S rRNA causes increased initiation at the UUG codon, which has been proposed to have caused redistribution of the ribosomal rotational equilibrium to the unrotated state due to uS19 LGH112‐114AAA substitution mutation, thereby underlying the importance of this region in start codon selection . Consistent with the suppression of UUG codon selection, the Gcn − phenotype associated with the eIF5 G31R mutant was suppressed by preventing upUUG codon recognition from the GCN4 transcript by the C1209U mutant (Fig.…”