Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase.
This work reveals that mutant forms of RNA methyltransferases that form 5-methylcytosine (m5C) have characteristics that may make them useful for biomacromolecular assembly. The experiments utilized bacterially expressed Trm4p, a tRNA methyltransferase cloned from Saccharomyces cerevisiae. Like DNA m5C methyltransferases, Trm4p mediates methylation using a covalent intermediate, which would allow Trm4p to be trapped as a stable protein-RNA complex when the substrate RNA contains a modified cytosine base such as 5-fluorocytosine. However, mutant forms of Trm4p are identified that fail to release RNA resulting in the formation of denaturant stable methyltransferase-RNA complexes that contain only natural nucleotides. The ability to form stable complexes with natural RNA gives these mutant forms of Trm4p greater potential versatility for biomacromolecule construction applications than the wild-type Trm4p enzyme or DNA methyltransferases for which the trapping of the covalent intermediate requires the presence of a nucleotide analogue at the site of modification.
Redman
Kent L
KL
Indiana University School of Medicine-Fort Wayne, 2101 Coliseum Boulevard East, Fort Wayne, Indiana 46805, USA. redman@ipfw.edu
D001483:Base Sequence; D003596:Cytosine; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010957:Plasmids; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012359:tRNA Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011506:Proteins; D012313:RNA; D003596:Cytosine; D012359:tRNA Methyltransferases