RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.
Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.
Nandakumar
Jayakrishnan
J
Molecular Biology and Structural Biology Programs, Sloan-Kettering Institute, New York, New York 10021, USA.
Ho
C Kiong
CK
Lima
Christopher D
CD
Shuman
Stewart
S
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011130:RNA Ligase (ATP) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D012331:RNA, Fungal; D014764:Viral Proteins; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2