Fate of the message-ribosome complex upon translation of termination signals

Abstract

A technique has been developed by which the translation of an f2 RNA molecule by a single ribosome can be monitored in an Escherichia coli S-30 extract. The extract is prepared by first incubating it with purified l-asparaginase II to remove all free asparagine and then with small amounts of sus 3 RNA to remove asparaginyl-tRNA. Addition of labeled phage RNA to a protein-synthesizing system containing the treated extract results in the formation of a rather stable phage RNA-ribosome-peptidyl-tRNA complex stopped at a site coding for an early asparagine residue in the coat protein. The addition of asparagine and other amino acids allows translation of the coat cistron to resume. Any further binding of ribosomes to the labeled RNA is prevented by either adding an excess of unlabeled phage RNA or making the reaction 70 μ m in aurintricarboxylic acid at the time of adding asparagine. Under these conditions, it is shown that aurintricarboxylic acid only prevents chain initiation and not chain propagation or termination of protein synthesis. Using this technique, it is shown that: (1) the rate of in vitro translation of the f2 phage coat cistron is 25 to 30 amino acids per minute; (2) translating the amber codon specifying site 6 of the coat protein as chain termination results in the complete release of the labeled phage RNA from the message-ribosome complex in not more than 0.75 minute; (3) translating the amber codon specifying site 70 of the coat protein as chain termination results in essentially the same rapid release of the RNA as for site 6; and (4) there is a rapid, but only a partial, release of the message when the natural chain terminating signal at the end of the coat cistron is translated. These results are discussed in terms of the mechanism of polarity and of the reading of intercistronic punctuation.