Formation and characterization of precise eucaryotic transcription complexes using a semisynthetic DNA template and...

An artificial template of defined sequence which supports specific in vitro initiation and elongation by yeast RNA polymerase II has been constructed. This template is a pBR322 derivative which contains a synthetic oligonucleotide inserted into the BamHI cloning site. The sequence of this oligonucleotide is such that when the plasmid is restricted with SacI the two ends obtained are identical. The addition of an oligodeoxycytidylate chain to the 3' hydroxy termini produces a DNA template, (poly dC-p+22), with the sequence: 3'(C)nTCGA-GAGTCTCCTA. . . . The underlined position denotes the beginning of the duplex region. When initiation is primed with the diribonucleotide GpC the predicted sequence of the transcript obtained is: 5'GCUCUCAGAGGAU. . . . Kinetic and product analyses indicate that a ternary complex containing a precise length of transcript can be produced which is subsequently resistant to heparin inactivation. Initiation can also be directed to a specific position dictated by a tri or tetraribonucleotide primer.