Abstract
We have established a new in vitro assay for translational termination. It consists of 70 S ribosomes bound to a synthetic RNA minimessenger via interaction with P-site binding fMet-tRNAfMet. If the A-site codon is a stop signal, release activity can be measured by quantifying hydrolyzed formylmethionine. Characteristics of this assay in terms of reaction time, ion concentration, release factor RF1 and RF2 concentration, and competition with A-site-decoding tRNA are discussed. The new assay shows that polypeptide chain release activity is directly dependent on the presence of a stop codon in the ribosomal A-site.
Highlights
We have established a new in vitro assay for translational termination
In complexes where the A-site was not occupied by a stop codon, fMet release was dependent on free stop triplet addition and high release factor concentration
Recognition of stop codons depends on participation of termination factors, which was initially suggested by Ganoza [39]
Summary
We have established a new in vitro assay for translational termination It consists of 70 S ribosomes bound to a synthetic RNA minimessenger via interaction with P-site binding fMet-tRNAfMet. If the A-site codon is a stop signal, release activity can be measured by quantifying hydrolyzed formylmethionine. The new assay shows that polypeptide chain release activity is directly dependent on the presence of a stop codon in the ribosomal A-site. Studies by immunomicroscopy [15] questioned the idea of release factors entering the ribosomal A-site like a tRNA. The study of the functional sites of release factors [19, 20] indicates that RF1 and RF2 might have a tRNA-like shape, spanning the codon recognition site and peptidyltransferase [21]. A tRNAlike shape for release factors has repeatedly been proposed [21, 24, 27]
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