title: Structure of the guanylyltransferase domain of human mRNA capping enzyme.
abstract: The enzyme guanylyltransferase (GTase) plays a central role in the three-step catalytic process of adding an (m7)GpppN cap cotranscriptionally to nascent mRNA (pre-mRNAs). The 5'-mRNA capping process is functionally and evolutionarily conserved from unicellular organisms to human. However, the GTases from viruses and yeast have low amino acid sequence identity (∼25%) with GTases from mammals that, in contrast, are highly conserved (∼98%). We have defined by limited proteolysis of human capping enzyme residues 229-567 as comprising the minimum enzymatically active human GTase (hGTase) domain and have determined the structure by X-ray crystallography. Seven related conformational states of hGTase exist in the crystal. The GTP-binding site is evolutionarily and structurally conserved. The positional variations of the oligonucleotide/oligosaccharide binding fold lid domain over the GTP-binding site provide snapshots of the opening and closing of the active site cleft through a swivel motion. The pattern of conserved surface residues in mammals, but not in yeast, supports the finding that the recognition of the capping apparatus by RNA polymerase II and associated transcription factors is highly conserved in mammals, and the mechanism may differ somewhat from that in yeast. The hGTase structure should help in the design of biochemical and molecular biology experiments to explore the proteinprotein and proteinRNA interactions that ensure regulated transcription of genes in humans and other mammals.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854, USA.
authors.forename: Chun
authors.initials: C
authors.lastname: Chu
authors.forename: Kalyan
authors.initials: K
authors.lastname: Das
authors.forename: James R
authors.initials: JR
authors.lastname: Tyminski
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Bauman
authors.forename: Rongjin
authors.initials: R
authors.lastname: Guan
authors.forename: Weihua
authors.initials: W
authors.lastname: Qiu
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Eddy
authors.initials: E
authors.lastname: Arnold
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Shatkin
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D000098556:mRNA Guanylyltransferases
chemical_list: D005656:Fungal Proteins; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D000098556:mRNA Guanylyltransferases