Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase.
The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.
Trippe
Ralf
R
Department of Biochemistry, Ruhr University Bochum, D-44780 Bochum, Germany.
Richly
Holger
H
Benecke
Bernd-Joachim
BJ
D001483:Base Sequence; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase