Deletion of a Conserved, Central Ribosomal Intersubunit RNA Bridge

Abstract

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Roles of helix H69 of 23S rRNA in translation initiation

“…Helix H69 contributes to the central intersubunit bridge B2a, loss of which shifts the 30S + 50S ⇌ 70S equilibrium strongly leftward (21). However, our LS experiments show that ΔH69 50S subunits are capable of docking to the 30SIC, with rate and amplitude comparable to the WT (Fig.…”

“…Thus, deletion of H69 traps the IC at the 70SICi stage and prevents the complex from entering the elongation phase. This provides a plausible explanation to the dominant lethal phenotype of H69 deletion in vivo (21), as ΔH69 50S subunits would trap ICs in a nonproductive form and prevent components from being accessed by WT 50S subunits.…”

“…Deletion of H69 (ΔH69, Fig. 1C) has been shown to cause dominant lethality in vivo and a severe subunit association defect in vitro that can only be partially rescued in the presence of mRNA, tRNA, and a high concentration of Mg 2+ (21). Despite these strong effects, ΔH69 ribosomes are fully competent for in vitro poly-Phe synthesis (21) and are only mildly affected in translocation (22).…”

“…1C) has been shown to cause dominant lethality in vivo and a severe subunit association defect in vitro that can only be partially rescued in the presence of mRNA, tRNA, and a high concentration of Mg 2+ (21). Despite these strong effects, ΔH69 ribosomes are fully competent for in vitro poly-Phe synthesis (21) and are only mildly affected in translocation (22). During subunit association, bridge B2a is among the earliest interactions formed between the two subunits (23), and H69 is in close proximity to the binding sites of initiator tRNA and IF3 (11,12).…”

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

“…In an effort to define the contribution of H69 to ribosome function, the properties of ribosomes from which the helix had been deleted were characterized (15). Although the ⌬H69 ribosomes displayed substantial defects in subunit association (and even exhibit ribosome recycling factor-independent recycling) and RF1-mediated catalysis, the particles were surprisingly active in poly-Phe synthesis and displayed only modest defects in accuracy during tRNA selection (the variant ribosomes incorporated ϳ2-fold less leucine on the poly-Uridine template).…”