JBC, Vol. 250, Issue 16, 6248-6255, Aug, 1975
In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities
E. K. Bikoff, B. F. LaRue and M. L. Gefter
We have shown that the synthesis of active su+III tRNATyr from a
phi80psu+III DNA template requires the action of four distinct enzymatic
activities. The first of these, DNA-dependent RNA polymerase, catalyzes the
formation of a large molecular weight transcript, initiating synthesis at a
specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr
structural gene and continuing at least 100 nucleotides beyond the 3'
terminus of the su+III tRNATyr sequence. The second required component,
designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to
function in tRNA synthesis. We have shown that this fraction contains an
endonuclease that together with DNA-dependent RNA polymerase is responsible
for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr
precursor is not itself the primary transcription product of the su+III
tRNATyr gene, but rather, it arises as a result of post-transcriptional
cleavage of a much larger transcript by the action of the nuclease present
in Fraction V. The third enzymatic activity required for synthesis of
active su+III tRNATyr is a ribonuclease (RNase P III) that specifically
catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr
precursor. The fourth activity required for synthesis of tRNA is a
previously identified endonuclease, RNase P, that specifically catalyzes
the removal of the 5' extra nucleotides from tRNA precursors. The
properties of RNase P purified according to the procedure developed in this
laboratory have been compared with those of the enzyme purified from
ribosomes according to the procedure described by Robertson et al.
(Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247,
5243-5251.).
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