All tRNA molecules contain, at the 3′ terminus, the identical-CCA sequence that is required for their acceptor and transfer functions. In prokaryotic cells, no discrete pattern has emerged. In laboratory strains of Escherichia coli, all tRNA genes sequenced encode the -CCA sequence, and mutant strains devoid of tRNA nucleotidyltransferase retain viability, although they grow slowly. The most convenient tRNA substrate for routine use during purification is commercial bakers' yeast tRNA. Generally, this material contains about 50% tRNA-CC and is active with tRNA nucleotidyltransferases from most sources. If more defined preparations of tRNA-CC, tRNA-C, or tRNA-N are required, they can be prepared by single or multiple cycles of periodate oxidation and alkaline phosphatase treatment of any tRNA. The filters are placed along the side of miniscintillation vials, dried under an infrared lamp for 10 minutes, and counted in a scintillation counter with 3 ml of scintillation fluid. Under certain conditions, tRNA nucleotidyltransferases can make errors in nucleotide incorporation, and these anomalous reactions have been used successfully for the synthesis of tRNA molecules with altered 3′ termini.
ASJC Scopus subject areas
- Molecular Biology