Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1.
Class-1 polypeptide chain release factors (RFs) play a key role in translation termination. Eukaryotic (eRF1) and archaeal class-1 RFs possess a highly conserved Asn-Ile-Lys-Ser (NIKS) tetrapeptide located at the N-terminal domain of human eRF1. In the three-dimensional structure, NIKS forms a loop between helices. The universal occurrence and exposed nature of this motif provoke the appearance of hypotheses postulating an essential role of this tetrapeptide in stop codon recognition and ribosome binding. To approach this problem experimentally, site-directed mutagenesis of the NIKS (positions 61-64) in human eRF1 and adjacent amino acids has been applied followed by determination of release activity and ribosome-binding capacity of mutants. Substitutions of Asn61 and Ile62 residues of the NIKS cause a decrease in the ability of eRF1 mutants to promote termination reaction in vitro, but to a different extent depending on the stop codon specificity, position, and nature of the substituting residues. This observation points to a possibility that Asn-Ile dipeptide modulates the specific recognition of the stop codons by eRF1. Some replacements at positions 60, 63, and 64 cause a negligible (if any) effect in contrast to what has been deduced from some current hypotheses predicting the structure of the termination codon recognition site in eRF1. Reduction in ribosome binding revealed for Ile62, Ser64, Arg65, and Arg68 mutants argues in favor of the essential role played by the right part of the NIKS loop in interaction with the ribosome, most probably with ribosomal RNA.
Frolova
Ludmila
L
Engelhardt Institute of Molecular Biology, Moscow, Russia.
Seit-Nebi
Alim
A
Kisselev
Lev
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry*; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012358:RNA, Transfer, Met / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003062:Codon; D017931:DNA Primers; C489002:ETF1 protein, human; D009842:Oligopeptides; D010454:Peptide Termination Factors; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet)