JBC, Vol. 250, Issue 16, 6240-6247, Aug, 1975
In vitro synthesis of transfer RNA. I. Purification of required components
E. K. Bikoff and M. L. Gefter
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.
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