A tripeptide 'anticodon' deciphers stop codons in messenger RNA.
The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.
Ito
K
K
Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Japan.
Uno
M
M
Nakamura
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000926:Anticodon / Q000235:genetics*; D001665:Binding Sites; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000502:physiology; D010454:Peptide Termination Factors / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics*
D000596:Amino Acids; D000926:Anticodon; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D010454:Peptide Termination Factors; D012333:RNA, Messenger; C041460:peptide chain termination release factor 2; C489005:prfA protein, E coli; C489006:prfB protein, E coli