Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

Abstract

Translation of the genetic code requires the accurate selection of elongation factor (EF)-TuGTPtRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G --A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA-namely, modulation of EF-Tu activity during translation.One of the most important clues to the functional importance of rRNA is its high phylogenetic sequence conservation. Now that a great many rRNA primary structures are available, spanning the phylogenetic spectrum, it is apparent that the nucleotide sequences of certain regions of these RNAs are essentially invariant in all organisms. This degree of conservation is most likely indicative of an indispensable functional role for these sequences in protein synthesis (1). Not even the well-known Shine-Dalgarno sequence (2) shares this degree of conservation, arguing that these universally conserved RNA elements must be involved in function at the most fundamental levels.Our knowledge of the roles of the most prominently conserved regions of rRNA supports this view (3). The sequences around positions 1400 and 1500 of the 16S-like rRNAs are the site of codon-anticodon interaction (4-6). In the 23S-like rRNAs, the central loop of domain V interacts with the other extremity of tRNA-the aminoacyl end-and is strongly implicated in the peptidyl transferase function (7-9). A highly conserved, 15-nucleotide hairpin loop (the "a-sarcin loop") in domain VI of 23S rRNA has been identified with elongation factor (EF)-Tu-and EF-G-dependent functions (10, 11). Its structural counterpart in 16S rRNA is the similarly conserved 16-nucleotide loop in the 530 region of 16S rRNA, whose functional role has proven more elusive.A growing body of evidence indicates that the 530 stemloop region plays an important role in tRNA selection in the ribosomal A site (reviewed in ref. (23). Detailed structure probing of mutant ribosomes revealed no evidence for perturbation of 16S rRNA structure or loss of interactions with ribosomal proteins, consistent with the idea that this mutation affects ribosomal function directly.In this study, we ch...

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

“…Another factor contributing to the defective behavior of the mutant might be a perturbation of the interactions of the factor with ribosomal elements required for GTPase activation following the codoninduced structural change. Such potentially involved EF-Tu-ribosome interactions have been characterized in the 530 loop of 16S RNA (Powers and Noller, 1993;Noller et al, 1996), in domain II of 23S rRNA (GTPaseassociated region; Rosendahl and Douthwaite, 1994) and in domain VI of 23S rRNA (ax-sarcin loop; Moazed et al, 1988;Tapprich and Dahlberg, 1990).…”

Drosophila melanogaster mitochondrial DNA: completion of the nucleotide sequence and evolutionary comparisons

“…T -A G -C that the defect involves a function related to EF-Tu (Powers & Noller, 1993). In addition, many of the nucleotides in the E. coliSSU rRNA flanking helix 49 that are protected by Asite and P-site tRNAs are conserved in the Drosophila 12s rRNA.…”

A Convergence of rRNA and mRNA Quality Control Pathways Revealed by Mechanistic Analysis of Nonfunctional rRNA Decay

“…Work from our laboratory has shown that rRNAs containing deleterious mutations in either the peptidyl transferase center of 25S rRNA or the decoding site of 18S rRNA are subject to a late-acting quality control system dubbed nonfunctional rRNA decay (NRD) (LaRiviere et al, 2006). In bacteria, the highly conserved bases A2451 and U2585 ( E. coli numbering) in the peptidyl transferase center and G530 and A1492 in the decoding site are all required for proper ribosome function, and mutations at any of these positions have dominant negative phenotypes (Green et al, 1997; Powers and Noller, 1990, 1993; Thompson et al, 2001; Yoshizawa et al, 1999; Youngman et al, 2004). In contrast, mutations introduced at the analogous positions in yeast are recessive due to degradation of the rRNAs containing them.…”