Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: A crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

Structures of Yeast 80S Ribosome-tRNA Complexes in the Rotated and Nonrotated Conformations

“…4A). Our structural model is consistent with results of biochemical experiments showing that the nucleotide in position −3 cross-links with G904 (Demeshkina et al, 2000) and S5 (Pisarev et al, 2006) in mammalian ribosomes.…”

Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

“…Cross-linking of mRNA to AA 1818-1819 has been detected with midrange nucleotide derivatives but not with "zero-length" cross-linkers: No cross-linking of AA 1818-1819 to thioU[ + 4] was observed in phased or unphased 80S complexes (Demeshkina et al 2000;Bulygin et al 2005). The equivalent prokaryotic nucleotides (AA 1492-1493 in T. Thermophilus) flip out upon binding of cognate aminoacyl tRNA to the A-site during elongation and interact with the minor groove of the first two base pairs of the base-paired codon-anticodon helix, thereby monitoring the fidelity of elongator tRNA selection (Ogle et al 2001).…”

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+…”