Cleavage of Escherichia coli tyrosine tRNA 2 in the S-region and its effects on the structure and function of the reconstituted molecules

Abstract

1. 1. Upon limited digestion of Escherichia coli tRNA Tyr 2 with ribonuclease T 1 in the presence of Mg 2+, splits occurred in the so-called S-region, and also in the TψC-loop: The resulting tRNA fragments were well separated from each other by means of column chromatography and their nucleotide compositions were characterized. 2. 2. Tyrosine-acceptor activity was restored only when the pG-containing fragment (Fragment D), which resulted from a cleavage at the S-region and the CCA-containing fragment (Fragment B) which is complementary to Fragment D, were combined. No tyrosine was accepted by either Fragment alone. For the maximum level of restoration, pre-incubation of the combined fragments in the presence of Mg 2+ was necessary before the activity assay. Another CCA-containing fragment (Fragment A) which is shorter than Fragment B as a result of split in the TψC-loop could not accept tyrosine when combined with Fragment D. However, a competition experiment showed that Fragment A could interact to form a complex with Fragment D but with less efficiency when compared with Fragment B. Thus, the pG- and CCA-terminal base-paired stem region by itself seemed to be sufficient for maintaining the A-D complex. 3. 3. Binding of the B-D complex to ribosomes was stimulated by poly (U 5, A) but the efficiency of the binding was less than 10 % when compared with that of native tyrosine tRNA 2. No stimulation of the binding was observed when the A-D complex was used. When poly (U, A, C) was used as messenger, nonspecific binding was observed even in the absence of ribosomes. The data are discussed in reference to the base composition of the synthetic messenger and the abnormal structure of the reconstituted tRNA molecules.