Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors

Abstract

In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universalcode ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124 -131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons. ciliated protozoa ͉ dual gene reporter system ͉ eukaryote class-1 polypeptide release factors ͉ interdomain and intradomain protein chimeras ͉ stop codon decoding I n the universal genetic code, three stop codons (UAA, UAG, and UGA) located at the termini of mRNA sequences are decoded at the termination step of translation by class-1 polypeptide release factors (RF) (reviewed in refs. 1-3). However, in organisms with variations in the genetic code, like ciliates, class-1 factors are able to decode only one or two stop codons with the remaining stop codon(s) reassigned to encode certain amino acids (for review, see refs. 4 and 5). The molecular mechanisms that restrict stop codon recognition are entirely unknown and represent major unresolved problems in molecular biology and genetics. In eukaryotes with the standard code, a single class-1 RF, designated eukaryote class-1 polypeptide release factor (eRF1), decodes all three stop codons. Stop codon decoding results in signal transduction from the small to the large ribosomal subunit leading to cleavage of peptidyl-tRNA at the peptidyl transferase center of the ribosome.The eRF1 protein family is highly conserved and similar to archaeal class-1 RFs but differs profoundly from bacterial class-1 RFs (6-8). The only known structural element common to all class-1 RFs is a univer...

Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

“…1 H, 13 C, and 15 N chemical shifts were assigned for $99% of the protein backbone resonances of the isolated N-domain. More than 85% of all the side chain 1 H, 13 C, and 15 N chemical shifts were also assigned.…”

“…1 H, 13 C, and 15 N chemical shifts were assigned for $99% of the protein backbone resonances of the isolated N-domain. More than 85% of all the side chain 1 H, 13 C, and 15 N chemical shifts were also assigned. The amide HN and 15 N signals of the residue Asn86 could not be observed in the 15 N-1 H HSQC spectra and the signals of residues Arg65, Glu103, and Thr133 had reduced intensities, probably due to fast exchange with water.…”

“…NOE distance restraints were determined from the [ 1 H, 13 C] NOESY and [ 1 H, 15 N] NOESY spectra measured at 298 K with a 100 ms mixing time. NOE peak volumes were calculated using the Sparky software.…”

“…32 Sequential backbone assignments and side chains assignments were obtained using the following 2D and 3D spectra: DQF-COSY, [ 13 15 N atoms of the protein backbone and for more than 90% of the side chains atoms. Backbone / and w dihedral angle restraints were determined from the chemical shift values of the backbone atoms 13 Ca, 13 Cb, 13 C 0 , 1 Ha, 1 HN, and 15 N using TALOSþ software. 34,35 Side chain dihedral angles v1 were obtained by the AngleSearch program.…”

“…More recently, the role of the N-domain has been confirmed unambiguously: chimeric eRF1s, in which the N-and MCdomains were derived from variant-code ciliate organisms 11,12 and human/yeast eRF1, respectively, exhibit ciliate stop codon decoding specificity in vitro. [13][14][15][16] Several hypotheses for the molecular mechanism of stop codon recognition by eRF1 have been suggested, 9,[17][18][19][20][21][22][23] but none satisfactorily explain the available experimental data which are somewhat contradictory.…”