Characterization of T7-specific Ribonucleic Acid Polymerase

Abstract

Abstract The major in vitro RNA transcripts synthesized by T7 RNA polymerase with T7 and T3 DNA templates have been resolved by electrophoresis on polyacrylamide gels. Six discrete size classes of T7 RNAs are found, designated I to VI. The apparent molecular weights, estimated from their electrophoretic mobilities, span a broad range, from 2 x 105 to 5 x 106. At least two minor RNA species, with molecular weights greater than 5 x 106, are also detected. The six major T7 RNA species are synthesized in approximately equimolar amounts, with the exception of species III, which is made in about twice this amount. It is likely that species III is a mixture of two RNAs (IIIa and IIIb) transcribed from separate regions of the T7 genome. Hence, the six major T7 RNA species are tentatively identified with seven late transcription units on the phage chromosome, which are read with equal efficiences. The six (or seven) transcription products are initiated independently with guanosine triphosphate at the 5' terminus, and are elongated at a rate of 230 nucleotides per s under standard in vitro conditions. When T7 RNA polymerase is used to transcribe T3 DNA, a single RNA transcript is found, with a molecular weight similar to that of T7 RNA species III. This suggests an explanation for the reduced rate of T3 RNA synthesis by T7 RNA polymerase in vitro, and implies that T3 promoter sites read by the T3 RNA polymerase are heterogeneous in nature.

Keywords

Affiliated Institutions

• University of California, Berkeley US

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