Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

“…This is consistent with the observation that clinically relevant aminoglycosides can be used to successfully treat bacterial infections in patients (Keeling and Bedwell 2005). Interestingly, the rdn15 mutation (identified as an A1754G mutation in 18S rRNA) was previously isolated as a suppressor of the conditional lethality associated with a yeast eRF1 mutation (sup45-R2 ts ) (Velichutina et al 2001). Thus, while this nonconserved nucleotide does not appear to contribute directly to the decoding of sense codons, it clearly influences the recognition and/or function of this mutant release factor.…”

“…Each repeat unit contains the genes for 5.8S, 18S, and 25S rRNAs that are transcribed by RNA polymerase I, as well as the 5S rRNA gene transcribed by RNA polymerase III. Previous studies have used a yeast strain in which the majority of the chromosomal rDNA repeats were deleted (Chernoff et al 1994;Velichutina et al 2001), with cell viability maintained by the expression of an rDNA repeat from a plasmid. However, a small number of copies of rDNA repeats remained in the genome of that strain, again resulting in mixed populations of wild-type and mutant ribosomes.…”

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

“…The pJD373 series of plasmids is based on the pJD211 series, and carry the C1495U mutation in helix 44 of 18S rDNA. This allele was previously shown to confer a recessive hygromycin-resistant phenotype on yeast cells (Velichutina et al 2001). Standard site-directed mutagenesis was used to generate the naturally occurring allelic variants of 5S rRNA found in yeast (RDN5-2 to RDN5-7) and the RDN5-Ooc and RDN5-Som hybrids of yeast and Xenopus 5S rRNAs, using the RDN5-1 allele cloned into pRS424, a high copy-number 2 m TRP1 vector (Christianson et al 1992) as the template.…”

Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1

“…The NIKS mutations that affect the eRF1 binding to the ribosome are Ile62Ala, Ile62Asn, Ser64Asp, and a double mutation Asn61Ser ϩ Ser64Asp (Fig+ 2A,B)+ Because Asn61Ser binds efficiently to the ribosome (Fig+ 2A) the effect of double mutation is due to Ser64Asp substitution+ A reduction in binding ability is probably responsible at least partly for the reduction of catalytic activity of these mutants+ It has been shown by a genetic approach that replacement of Arg65 for cysteine residue in yeast Sup45p later attributed to eRF1 (Frolova et al+, 1994) confers an omnipotent suppressor phenotype to the mutant Sup45p/eRF1 (Mironova et al+, 1986)+ At that time, these data were not explained, but now they are considered in view of potential location of the TCRS near this position (Song et al+, 2000)+ Our data (Fig+ 2B) provide evidence that, in fact, this position is essential both for ribosome binding and stop codon recognition (Arg65 in yeast eRF1 corresponds to Arg68 in human eRF1; see Fig+ 1A)+ However, the genetic approach does not allow us to discriminate between omnipotent suppression caused by reduced eRF1 binding to the ribosome and the reduction of catalytic activity caused by distortion of the TCRS or peptidyl-tRNA interaction site+ Numerous biochemical, structural, and genetic data point to a functional role for rRNA in tRNA selection (see Green & Noller, 1997)+ The decoding domain of 16S rRNA formed by helices 18, 24, 27, 34, and 44 affects the translational accuracy (Lodmell & Dahlberg, 1997;O'Connor et al+, 1997;Pagel et al+, 1997)+ Escherichia coli 16S rRNA mutations cause defects in translation termination (Arkov et al+, 1998) In eukaryotes, A1823 and A1824 in human 18S rRNA equivalent to E. coli G1491 and A1492, respectively, cross-react with the first position of the codon located at the A site (Demeshkina et al+, 2000)+ Mutations in the 18S rRNA affect fidelity of the stop codon decoding (Velichutina et al+, 2001, and references therein)+ Collectively, all these data point to the involvement of small rRNA sequences in codon-anticodon and stop codon-eRF1 interactions+ Coexistence of the elements of TCRS and RBS within the NIKS subdomain is entirely consistent with the close proximity of mRNA and small rRNA nucleotides at the A site+ Amino acids at positions 64, 65, and 68 of human eRF1 that affect the ribosome binding properties of eRF1 in the absence of mRNA and tRNA (Fig+ 2B) could interact with amino acid residues at positions 1492, 1493, and 530 of 18S rRNA (numeration as in E. coli 16S rRNA)+ Other nucleotides of 18S rRNA could be also implicated in this interaction+ It means, that RBS may in fact be in close vicinity toward the TCRS or even overlap and these sites may be concomitantly affected by a single mutation+…”