121
4.9.2 RNA Ligases
They are ATP-dependent enzymes that can catalyze the ligation of RNA molecules
via phosphodiester bonds. RNA ligases have been found in organisms of all major
phyla, where they serve important cellular functions like RNA splicing, editing, and
repair. RNA ligation occurs by the joining of 3′-hydroxyl RNA termini of one fragment to the 5′-phosphate termini of the adjacent fragment, through three nucleotidyl
transfer steps. The initial step involves the reaction between RNA ligase and ATP to
produce an intermediate covalent ligase-(lysyl-N)-AMP and pyrophosphate as
products. In the next step, the bound AMP is transferred from ligase-(lysyl-N)-AMP
to the 5′-phosphate termini of RNA to form an RNA-adenylate intermediate
(AppRNA) and in the final step, RNA ligase catalyze a nucleophilic attack on
AppRNA end by 3′-OH of the other RNA strand, releasing AMP and forming a
phosphodiester bond that joins the two RNA strands (Ho et al. 2004).
4.9.2.1 T4 RNA Ligase
T4 RNA ligase, isolated from E. coli cells infected with T4 phage, is the most
widely used and the best characterized of RNA ligases in molecular biology. This
48 kDa monomeric enzyme requires ATP as a cofactor to catalyze the ligation of
ssRNA, ssDNA, and polynucleotides to RNA molecules. T4 RNA ligase is used in
oligonucleotide synthesis to ligate oligonucleotide adaptors to cDNA, 5′ nucleotide
modifications of nucleic acids, primer extension for PCR, and for circularizing
RNA and DNA molecules. It can also be used for linking oligonucleotide adaptors
to 5′ termini of mRNA for techniques like RLM-RACE. Another RNA ligase has
now been identified in bacteriophage T4 which has been named T4 RNA ligase 2 or
T4 Rnl-2. It has the ability to catalyze both intramolecular and intermolecular RNA
strand ligations. T4 RNA ligase 2 has higher efficiency in nick sealing of dsRNA
than joining ssRNA fragment ends (Ho et al. 2004). A truncated form of T4 RNA
ligase 2 composed of only first 249 amino acids is ATP independent and lacks 5′
termini adenylation activity. It requires a pre-adenylated substrate for ligation reaction and is used in cloning of small RNAs because it restricts the background formation of circles and multimers of RNAs.
4.9.2.2 Thermostable RNA Ligases
Thermostable RNA ligases are sought after in molecular biology for use in techniques where the secondary structure of RNA becomes a constraint in ligation
experiments, which can be resolved by applying higher temperature. Such enzymes
have been isolated from a thermophilic archaebacteria, Methanobacterium thermoautotrophicum, and from two thermophilic bacteriophages TS2126 and RM378 that
infect the thermophilic eubacteria Thermus scotoductus and Rhodothermus marinus, respectively (Blondal et al. 2003, 2005).
4 Enzymes as Molecular Tools
4.9.2 RNA Ligases
They are ATP-dependent enzymes that can catalyze the ligation of RNA molecules
via phosphodiester bonds. RNA ligases have been found in organisms of all major
phyla, where they serve important cellular functions like RNA splicing, editing, and
repair. RNA ligation occurs by the joining of 3′-hydroxyl RNA termini of one fragment to the 5′-phosphate termini of the adjacent fragment, through three nucleotidyl
transfer steps. The initial step involves the reaction between RNA ligase and ATP to
produce an intermediate covalent ligase-(lysyl-N)-AMP and pyrophosphate as
products. In the next step, the bound AMP is transferred from ligase-(lysyl-N)-AMP
to the 5′-phosphate termini of RNA to form an RNA-adenylate intermediate
(AppRNA) and in the final step, RNA ligase catalyze a nucleophilic attack on
AppRNA end by 3′-OH of the other RNA strand, releasing AMP and forming a
phosphodiester bond that joins the two RNA strands (Ho et al. 2004).
4.9.2.1 T4 RNA Ligase
T4 RNA ligase, isolated from E. coli cells infected with T4 phage, is the most
widely used and the best characterized of RNA ligases in molecular biology. This
48 kDa monomeric enzyme requires ATP as a cofactor to catalyze the ligation of
ssRNA, ssDNA, and polynucleotides to RNA molecules. T4 RNA ligase is used in
oligonucleotide synthesis to ligate oligonucleotide adaptors to cDNA, 5′ nucleotide
modifications of nucleic acids, primer extension for PCR, and for circularizing
RNA and DNA molecules. It can also be used for linking oligonucleotide adaptors
to 5′ termini of mRNA for techniques like RLM-RACE. Another RNA ligase has
now been identified in bacteriophage T4 which has been named T4 RNA ligase 2 or
T4 Rnl-2. It has the ability to catalyze both intramolecular and intermolecular RNA
strand ligations. T4 RNA ligase 2 has higher efficiency in nick sealing of dsRNA
than joining ssRNA fragment ends (Ho et al. 2004). A truncated form of T4 RNA
ligase 2 composed of only first 249 amino acids is ATP independent and lacks 5′
termini adenylation activity. It requires a pre-adenylated substrate for ligation reaction and is used in cloning of small RNAs because it restricts the background formation of circles and multimers of RNAs.
4.9.2.2 Thermostable RNA Ligases
Thermostable RNA ligases are sought after in molecular biology for use in techniques where the secondary structure of RNA becomes a constraint in ligation
experiments, which can be resolved by applying higher temperature. Such enzymes
have been isolated from a thermophilic archaebacteria, Methanobacterium thermoautotrophicum, and from two thermophilic bacteriophages TS2126 and RM378 that
infect the thermophilic eubacteria Thermus scotoductus and Rhodothermus marinus, respectively (Blondal et al. 2003, 2005).
4 Enzymes as Molecular Tools