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Nucleic Acids Research, 1980, Vol. 8, No. 18 4111-4124
© 1980

MOLECULAR BIOLOGY

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

András Simoncsits

Institute of Genetics, Biological Research Center Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary

Received July 18, 1980. Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from {gamma}-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' -(ß-32P)-pyrophosphate (pGFormula) is prepared from {gamma}-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGFormula 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'-(ß-32P)-pyrophosphate labelled molecule.


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