Transcription initiation in eukaryotes: analysis of heterologous in vitro systems utilizing components from mammalian...

The properties of heterologous in vitro transcription systems utilizing components from mammalian and yeast cells have been investigated. Purified yeast RNA polymerase II, when supplemented with a full complement of mammalian transcription factors, does not promote specific transcription initiation on cloned mammalian class II genes. Similarly, a complete mammalian transcription system does not initiate specific transcription on cloned yeast class II genes. These results indicate evolutionary divergence in function of yeast and mammalian class II genes and the associated transcription apparatus. The functional differences observed in this study are corroborated by previously reported structural differences between yeast and mammalian RNA polymerase II and class II genes. In contrast, the mechanism of eukaryotic class III gene transcription appears to be evolutionarily conserved. Thus, a mammalian transcription extract specifically transcribes the cloned yeast 5S rRNA gene. This system synthesizes without processing the 130 base 5S rRNA precursor, and this primary transcript may be processed to the mature 120 base RNA using a partially purified yeast processing activity. An homologous yeast class III transcription system has also been developed. This yeast system contains all components necessary for proper synthesis, processing and splicing of yeast tRNA precursors. Using the homologous yeast transcription system, a template in which the 5' flanking region and first 5 base pairs of the 5S rRNA gene had been deleted is utilized to synthesize a 120 base transcript, but the efficiency of transcription is reduced to about 10% that of the wild type gene. Thus, the yeast 5S rRNA gene has an internal transcription control region, but the immediate 5' flanking sequence have effects on the level of transcription.