DNA-directed cell-free synthesis of biologically active transfer RNA: su + 3 tyrosyl-tRNA

Biologically active su(+) (III) tyrosyl-tRNA has been synthesized in a DNA-directed cell-free system from Escerichia coli. Such a system should be most useful for studying the mechanism of tRNA synthesis. This tRNA is capable of suppressing amber mutations in the gene coding for beta-galactosidase (EC 3.2.1.23) and, therefore, must be capable of being charged and transferring amino acids. A 4-fold stimulation in the activity of the tRNA formed de novo is obtained with isopentenyl pyrophosphate, a compound involved in the post-transcriptional acylation of an adenine base adjacent to the anticodon. It has been suggested elsewhere that formation of RNA subject to stringent control may be inhibited by guanosine tetraphosphate (ppGpp). However, guanosine tetraphosphate did not affect the synthesis of su(+)III tyrosyl-tRNA, even though the synthesis of this tRNA is subject to stringent control.