The role of the universally conserved A2450–C2063 base pair in the ribosomal peptidyl transferase center

Abstract

Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450–C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450–C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.

Molecular dynamics investigation of a mechanism of allosteric signal transmission in ribosomes

“…Therein, nucleotide residue G2061 directly contacts C2063, whereas A2062 is either taken outside ("open" conformation) or located nearby G2061, but not C2063 ("closed" conformation). Nucleotide residues G2061 and C2063 belong to the so called "outer shell" of the PTC [55] and participate in structuring of A2451, one of the key functional nucleotide residues of the ribosomal PTC [56,57], whereas their site directed mutagenesis dramatically affected activity of the ribosome [58,59]. Thus, this suggests that gradual shifting of C2063 relative to its position in the starting structure as well as complete loss of its contacts with G2061 by 80 nsec can also affect efficacy of the peptidyl transferase (PT) reaction.…”

Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development

“…In the PTC there is just 1 nt difference: U3780C. But even a single difference in such a conserved region is worth noticing, knowing that in the prokaryote PTC-equivalent, individual nucleotides have specific roles in the translation process and some mutations result in a lethal phenotype (Beringer 2008;Polacek and Mankin 2008;Long et al 2009Long et al , 2010Yang et al 2009;Chirkova et al 2010).…”

Atomic Mutagenesis of the Ribosome: Towards a Molecular Understanding of Translation

“…Binding of a class I RF to the A-site of the ribosome results in the hydrolysis of the tional GTPases, [29,30] and tRNA translocation. [31] One of the most surprising results of our recent research was the finding that a single rRNA backbone group, namely the ribose 2'-OH of the inner core PTC residue A2451, rather than any of the universally conserved nucleobases is crucial for peptide bond synthesis (Fig. 2).…”

“…Employing the atomic mutagenesis approach we were able to identify the importance of a non-Watson-Crick base pair between the two active site residues A2450-C2063 for tRNA translocation. [31] In addition, we identified a functional group at an adenosine at position A2660 of the 23S rRNA to be pivotal for activating the GTPase activity of EF-G (Fig. 2).…”