In vitro synthesis of transfer RNA. I. Purification of required components.
We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III 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 su++III 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 su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable 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 vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 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.
D002850:Chromatography, Gel; D003090:Coliphages / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005696:Galactosidases / Q000096:biosynthesis; D007700:Kinetics; D009154:Mutation; D012329:RNA, Bacterial / Q000096:biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014443:Tyrosine
D012329:RNA, Bacterial; D012367:RNA, Viral; D014443:Tyrosine; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases; D005696:Galactosidases