Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II.
5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.
Yue
Z
Z
Center for Advanced Biotechnology and Medicine, Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854-5638, USA.
Maldonado
E
E
Pillutla
R
R
Cho
H
H
Reinberg
D
D
Shatkin
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D000098556:mRNA Guanylyltransferases
D009713:Nucleotidyltransferases; D012319:RNA Polymerase II; C020842:guanylyltransferase; D000098556:mRNA Guanylyltransferases