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(Received for publication, July 14, 1997, and in revised form, September 17, 1997)
From the Department of Biological Sciences, Columbia University,
New York, New York 10027
The poly(A) tail of a mammalian mRNA is
generated by endonucleolytic cleavage and poly(A) addition. Previous
studies conducted with nuclear extracts suggested an ATP requirement
for the cleavage step. We have reexamined the cofactor requirement,
initially with the SV40 late pre-mRNA, which requires for cleavage
four protein factors, cleavage and polyadenylation specificity factor,
cleavage stimulation factor, cleavage factor I, and cleavage factor II. Using highly purified preparations of these factors, which lacked detectable creatine phosphokinase and ATPase activities, creatine phosphate (CP) was, surprisingly, found to be sufficient to promote efficient cleavage. Although other phosphate compounds substituted poorly or not at all for CP, another phosphoguanidine, arginine phosphate, was fully functional. Notably, ATP was neither necessary nor
sufficient, and could in fact inhibit the reaction. Treatment of the
purified factors with hexokinase plus glucose (to deplete any
contaminating ATP) was without effect, as was addition of EDTA. Using
32P-labeled CP, we found that neither hydrolysis of
CP nor phosphate transfer from CP occurred during the cleavage
reaction. CP also allowed cleavage of the adenovirus 2 L3 pre-mRNA.
However, in this case, ATP both enhanced the reaction and influenced
the precise site of cleavage, perhaps reflecting the requirement of
poly(A) polymerase for cleavage of this RNA. These results indicate
that ATP is not essential for 3
Volume 272, Number 47,
Issue of November 21, 1997
pp. 29636-29642
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Creatine Phosphate, Not ATP, Is Required for 3
End Cleavage of
Mammalian Pre-mRNA in Vitro
pre-mRNA cleavage and that CP
or a related compound can function as a necessary
cofactor.
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