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Nucleic Acids Research, 1980, Vol. 8, No. 22 5223-5232
© 1980

CHEMISTRY

Selective 32P-labelling of individual species in a total tRNA population

Cinzia Traboni+, Riccardo Cortese{dagger} and Francesco Salvatore+

+Istituto di Chimica Biologica, II Facoltà di Medicina, University of Naples Naples, Italy {dagger}European Molecular Biology Laboratory Heidelberg, GFR

Received September 16, 1980. A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E.coli) is incubated in the presence of a crude aminoacyl-tRMA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by ß-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.


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