Purification and In Vitro Transcription of a Transfer RNA Gene

Purification and In Vitro Transcription of a Transfer RNA Gene

Violet Daniel*,
Jacques S. Beckmann*,
Sara Sarid†,
Jacob I. Grimberg*,
Max Herzberg‡, and
Uriel Z. Littauer*

^*Department of Biochemistry, The Weizmann Institute of Science, Rehovot, Israel
^†Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel
^‡Department of Biological Ultrastructure, The Weizmann Institute of Science, Rehovot, Israel

Abstract

A gene specifying tyrosine transfer RNA has been purified and transcribed in vitro. The purification procedure made use of two specialized transducing phages carrying the tRNA^Tyr gene of Escherichia coli inserted into their DNA in opposite orientations. The separated heavy strands of the two phages were annealed and the single-stranded tails of the resulting hybrid were removed by digestion with Neurospora endonuclease. The size of the purified double-stranded structures was determined by electron microscopy. These isolated duplexes served as template for the in vitro transcription of tRNA^Tyr-like molecules.