Mutational Analysis of Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase I, a DNA-Dependent ATPase of the DExH Box Family

AUTOR(ES)

Martins, Alexandra

FONTE

American Society for Microbiology

RESUMO

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.

Documentos Relacionados

• Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.
• Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.
• Genetic evidence for involvement of vaccinia virus DNA-dependent ATPase I in intermediate and late gene expression.
• DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.
• Properties of a DNA-dependent ATPase from rat mitochondria.