Mutations of highly conserved bases in the peptidyltransferase center induce compensatory rearrangements in yeast ribosomes

Abstract

Molecular dynamics simulation identified three highly conserved rRNA bases in the large subunit of the ribosome that form a three-dimensional (3D) ''gate'' that induces pausing of the aa-tRNA acceptor stem during accommodation into the A-site. A nearby fourth base contacting the ''tryptophan finger'' of yeast protein L3, which is involved in the coordinating elongation factor recruitment to the ribosome with peptidyltransfer, is also implicated in this process. To better understand the functional importance of these bases, single base substitutions as well as deletions at all four positions were constructed and expressed as the sole forms of ribosomes in yeast Saccharomyces cerevisiae. None of the mutants had strong effects on cell growth, translational fidelity, or on the interactions between ribosomes and tRNAs. However, the mutants did promote strong effects on cell growth in the presence of translational inhibitors, and differences in viability between yeast and Escherichia coli mutants at homologous positions suggest new targets for antibacterial therapeutics. Mutant ribosomes also promoted changes in 25S rRNA structure, all localized to the core of peptidyltransferase center (i.e., the proto-ribosome area). We suggest that a certain degree of structural plasticity is built into the ribosome, enabling it to ensure accurate translation of the genetic code while providing it with the flexibility to adapt and evolve.

Simulating movement of tRNA through the ribosome during hybrid-state formation

“…The hybrid corridor is analogous to the accommodation corridor that we previously identified, 46 for which the constituent molecular interactions have been experimental corroborated. [65][66][67][68][69] …”

Selenocysteine Insertion Sequence (SECIS)-binding Protein 2 Alters Conformational Dynamics of Residues Involved in tRNA Accommodation in 80 S Ribosomes

“…A computer simulation of the accommodation pathway revealed that nucleotide U2473 (E. coli numbering/mouse U4071) in H89 makes direct contacts with the elbow region of the aa-tRNA in the early stages of the A/T to A/A transition and continuously associates with the backbone of the aa-tRNA throughout this process (27). Yet another simulation showed that H89 is displaced during accommodation, and it was suggested that residues in this helix can acquire alternative conformations for different aa-tRNAs (36,37). Comparison of x-ray structures of the 70 S ribosome with cognate or near cognate tRNA reveal a network of rRNA helices (including H89) and ribosomal protein contacts that stabilize the elbow of cognate tRNA in the A/A state (38).…”

Non-Stressful Death of 23S rRNA Mutant G2061C Defective in Puromycin Reaction

“…1 Many mutants of the peptidyl transferase region of ribosome have been reported in the literature. [1][2][3][4][5][6][7][8] The most widespread phenotype was reduction in puromycin reactivity, while inability to catalyze the peptidyl transferase reaction involving natural tRNA substrates was scarce. 1 Nevertheless, the majority of those mutations were lethal when tested in vivo.…”