The Journal of Biological Chemistry

We have described an in vitro system in which active s&r tRNATyr is synthesized from a @Ops&r DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes s&r tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the S&I tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detectable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3’ terminus of the tRNA precursor. In the absence of this fraction, the in vifro syntbesized s&r tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal -CCAon 3’ terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5’ extra nucleotides present on tRNA precursors.