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Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1
Ludmila Frolova1, Alim Seit-Nebi, Lev Kisselev
1Engelhardt Institute of Molecular Biology, Moscow, Russia.
RNA (New York, N.Y.)
|March 26, 2002
Summary
Class-1 polypeptide chain release factors (RFs) are crucial for translation termination. Mutations in the conserved Asn-Ile-Lys-Ser (NIKS) motif of human eRF1 reveal its role in stop codon recognition and ribosome binding.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Class-1 polypeptide chain release factors (RFs) mediate translation termination.
- Eukaryotic and archaeal class-1 RFs share a conserved Asn-Ile-Lys-Ser (NIKS) motif.
- The NIKS motif's conserved and exposed nature suggests a role in stop codon recognition and ribosome binding.
Purpose of the Study:
- To investigate the role of the NIKS tetrapeptide in human eRF1 function.
- To determine the impact of NIKS mutations on stop codon recognition and ribosome binding.
Main Methods:
- Site-directed mutagenesis of the NIKS motif (residues 61-64) and adjacent amino acids in human eRF1.
- In vitro determination of release activity and ribosome-binding capacity of generated mutants.
Main Results:
- Substitutions at Asn61 and Ile62 reduced termination activity, suggesting the Asn-Ile dipeptide modulates stop codon specificity.
- Mutations at positions 60, 63, and 64 had minimal impact, challenging existing hypotheses.
- Mutations in Ile62, Ser64, Arg65, and Arg68 decreased ribosome binding, indicating the NIKS loop's right side is crucial for ribosome interaction.
Conclusions:
- The Asn-Ile dipeptide within the NIKS motif plays a role in specific stop codon recognition by eRF1.
- The right portion of the NIKS loop is essential for eRF1 interaction with the ribosome, likely via ribosomal RNA.