3′ Terminal labelling of RNA with β-32P-pyrophosphate group and its application to the sequence analysis of 5S RNA from Streptomyces griseus

摘要

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from γ-32P-ATP to the 3′ -0H of tRNA, generating a strictly terminal label at its 3′ end. Using yeast tRNAphe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5′ -phosphate 3′ -(β-32P)-pyrophosphate (pG) is prepared from γ-32P-ATP and GMP by nucleotide pyrophosphate transferase. pG is then ligated to the 3′ end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3′-(β-32P)-pyrophosphate labelled molecule.