Journal: Nucleic Acids Research

Article Title: The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site

doi: 10.1093/nar/gkm1172

Figure Lengend Snippet: Overall structure of the M. jannaschii Nep1-protein and quality of the data. ( A ) View of the Nep1-dimer in the asymmetric unit. Subunit A is shown in orange and subunit B is shown in blue. The bound co-factor S -adenosylhomocysteine (SAH) is shown as a stick model. ( B ) Typical example for the quality of the experimental 2 F o - F c σ A -weighted electron density map at the 1.5 σ-level for the Nep1 bound to SAH. Shown are residues G53-I57.

Article Snippet: The gene encoding the full-length M. jannaschii Nep1-protein (aa1-205) was amplified by PCR from M. jannaschii genomic DNA (ATCC 43967D-5) with the appropriate primers and subcloned into the pET11a (Novagen, Madison) overexpression vector.

Techniques:

Journal: Nucleic Acids Research

Article Title: The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site

doi: 10.1093/nar/gkm1172

Figure Lengend Snippet: Fold of the Nep1-monomer. ( A ) Two views of the Nep1-monomer with the color gradient starting in blue at the N-terminus and changing to red at the C-terminus. The N- and C-terminus and the secondary structure elements are labelled. ( B ) Cartoon of the secondary structure of Nep1. α-helices are presented by circles and β-sheets are presented by triangles. The five-stranded parallel β-sheet typical for the fold of the SPOUT-class of methyl transferases corresponds to β-sheets β1, β2 and β5, β6 and β7. The structural elements that are part of the common core of SPOUT-class methyltransferase domains are highlighted in red. The β–α–β insertion element is encircled with a broken line. ( C ) ClustalW-alignment of the sequences of Nep1 from M. jannaschii (Mja), S. cerevisiae (yeast) and H. sapiens (human). Identical residues are highlighted in red, conservative replacements are indicated in green. A cartoon of the secondary structure is shown above the sequence alignment.

Article Snippet: The gene encoding the full-length M. jannaschii Nep1-protein (aa1-205) was amplified by PCR from M. jannaschii genomic DNA (ATCC 43967D-5) with the appropriate primers and subcloned into the pET11a (Novagen, Madison) overexpression vector.

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site

doi: 10.1093/nar/gkm1172

Figure Lengend Snippet: Electrostatic surface potential representation of the NepI dimer and possible RNA-binding amino acid residues. ( A ) Cartoon of the Nep1 dimer structure with the side chains of the four conserved arginines shown in a ball and stick-representation (top) and electrostatic surface potential map (bottom) in the same orientation. The approximate locations of the side chains of Arg 54, Arg 95, Arg 98 and Arg 102 and of the SAH-binding site are indicated by arrows. Amino acid residues from different monomers are differentiated by an asterisk. ( B ) Yeast three-hybrid experiments with the yeast Nep1-protein mutants. The arginine side chains equivalent to Arg 54 (Arg 88), Arg 95 (Arg 129), Arg 98 (Arg 132) and Arg 102 (Arg 136) of the M. jannaschii protein were mutated to alanine in yeast Nep1. The yeast numbering is given in brackets. Whereas the wild-type yeast protein binds to RNAs containing either one (pIII/UUCAAC) or three copies (pIII/3×UUCAAC) of the RNA-motif 5′-UUCAAC-3′ as indicated by significant β-galactosidase activity (given in Miller units, U/mg) but not to a control (pIII/MS2-2), the RNA-binding activity of all four mutants is strongly reduced.

Article Snippet: The gene encoding the full-length M. jannaschii Nep1-protein (aa1-205) was amplified by PCR from M. jannaschii genomic DNA (ATCC 43967D-5) with the appropriate primers and subcloned into the pET11a (Novagen, Madison) overexpression vector.

Techniques: RNA Binding Assay, Binding Assay, Activity Assay