EUKARYOTIC RELEASE FACTOR-1 (ERF1) ABOLISHES READTHROUGH AND COMPETESWITH SUPPRESSOR TRANSFER-RNAS AT ALL 3 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 fra me (ORF) of mRNAs depends on the availability of suppressor tRNA(s) an d the efficiency of termination in cells, Consequently, the yield of r eadthrough products can be used as a measure of the activity of polype ptide 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 fur ovirus is required for virus replication. Constructs harbouring this s uppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in v itro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukar yotic RF1 (eRF1). Thus, eRF1 is functional towards all three terminati on codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A si te. These results are consistent with a crucial role of eRF1 in transl ation termination and forms the essence of an in vitro assay for RF ac tivity based on the abolishment of readthrough by eRF1.