Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.
It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.
Drugeon
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G
Institut Jacques Monod, 2 Place Jussieu-Tour 43, 75251 Paris Cedex 05, France.
Jean-Jean
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O
Frolova
L
L
Le Goff
X
X
Philippe
M
M
Kisselev
L
L
Haenni
A L
AL
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010454:Peptide Termination Factors / Q000302:isolation & purification / Q000378:metabolism*; D010942:Plant Viruses / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014675:Vegetables / Q000821:virology; D014779:Virus Replication; D029867:Xenopus Proteins; D014982:Xenopus laevis
D003062:Codon; D017931:DNA Primers; C489002:ETF1 protein, human; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C489001:SUP45 protein, Xenopus; D029867:Xenopus Proteins; C489005:prfA protein, E coli; D012343:RNA, Transfer