A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.
A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.
Wilusz
J
J
Howard Hughes Medical Institute, Department of Biology, Princeton University, New Jersey 08544.
Shenk
T
T
Takagaki
Y
Y
Manley
J L
JL
D001483:Base Sequence; D002474:Cell-Free System; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000502:physiology*; D011061:Poly A / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012261:Ribonucleoproteins / Q000502:physiology* / Q000528:radiation effects / Q000648:ultrastructure; D014466:Ultraviolet Rays
D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D011061:Poly A