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Structure-function analysis of T4 RNA ligase 2.
Shenmin Yin1, C Kiong Ho, Stewart Shuman
1Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
The Journal of Biological Chemistry
|March 4, 2003
Summary
Bacteriophage T4 RNA ligase 2 (Rnl2) uses essential amino acids in conserved motifs for strand joining. Mutagenesis reveals shared active site features with DNA ligases and mRNA capping enzymes.
Area of Science:
- Molecular Biology
- Enzymology
- Structural Biology
Background:
- Bacteriophage T4 RNA ligase 2 (Rnl2) is a model for a polynucleotide ligase family.
- This family includes trypanosome RNA-editing ligases and viral/archaeal RNA ligases.
Purpose of the Study:
- To analyze essential amino acids in Rnl2 for strand joining.
- To determine structure-activity relationships and mutational effects on ligase function.
Main Methods:
- Alanine scanning mutagenesis to identify essential amino acids.
- Conservative amino acid substitutions to probe structure-activity.
- Analysis of adenylylation and phosphodiester bond formation steps.
Main Results:
- Twelve individual amino acids were identified as essential for strand joining.
- Essential residues are located in conserved nucleotidyl transferase motifs.
- Mutagenesis confirmed shared active site architecture with DNA ligases and mRNA capping enzymes.
Conclusions:
- Rnl2 and related ligases share a common active site architecture.
- Specific residues (Glu34, Asn40) flanking the active site lysine (Lys35) are unique to Rnl2-like ligases.