Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation.
To study the mechanism and factors required to form the 3' ends of polyadenylated mRNAs, we have fractionated HeLa cell nuclear extracts carrying out the normally coupled cleavage and polyadenylation reactions. Each reaction is catalyzed by a distinct, separable activity. The partially purified cleavage enzyme (at least 360,000 MW) retained the specificity displayed in nuclear extracts, since substitutions in the AAUAAA signal sequence inhibited cleavage. In contrast, the fractionated poly(A) polymerase (300,000 MW) lost all specificity. When fractions containing the cleavage and polyadenylation activities were mixed, the efficiency and specificity of the polyadenylation reaction were restored. Interestingly, the cleavage activity by itself functioned well on only one of four precursor RNAs tested. However, when mixed with the poly(A) polymerase-containing fraction, the cleavage activity processed the four precursors with comparable efficiencies.
Takagaki
Y
Y
Department of Biological Sciences, Columbia University, New York, New York 10027.
Ryner
L C
LC
Manley
J L
JL
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D013379:Substrate Specificity
D012322:RNA Precursors; D008345:Manganese; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase; D008274:Magnesium