STUDIES ON VALYL-TRNA SYNTHETASE AND TRNAVAL FROM ESCHERICHIA COLI .2. INTERACTION BETWEEN VALYL-TRNA SYNTHETASE AND VALINE ACCEPTOR TRNA

Complexes composed of equimolar amounts of enzyme and tRNA were isolated by sucrose gradient centrifugation. The same complex was also detected by the membrane-binding assay developed by Yarus & Berg (1967). An affinity constant of 108 liters/mole was measured at pH 5.5 and a single binding site was found per enzyme molecule. tRNAVal acylated, deacylated, or lacking its terminal adenosine bind to the enzyme with similar affinities. The presence of Val-ol-AMP, a competitive analogue of valyl-AMP, increased the preference for unacylated versus acylated tRNA binding by a factor of only 1.5, without a marked change of the affinity constants. The fact that enzyme and tRNA can be associated in a specific complex is illustrated also by the protection of tRNAVal against endonuclease attack. A resistant core resulting from partial digestion of an enzyme tRNA complex is found in association with the enzyme after sucrose gradient centrifugation. The 4-thiouridine-containing sequence (Yaniv & Barrell, 1969) is one of the preferentially protected regions in tRNAVal. © 1969.