HeLa cell RNA polymerase III transcription factors. Functional characterization of a fraction identified by its activity in a second template rescue assay.

Abstract

That stable transcription complexes are formed in HeLa cell RNA polymerase III transcription extracts (Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173) can be shown in order of template addition experiments with DNAs coding for adenovirus 2 VA I RNA or one of the Bombyx mori tRNAAla2 genes. Using this property of the HeLa extracts in a "second template rescue assay" has allowed us to partially purify protein components involved in stable transcription complex formation. Two fractions, called transcription fraction X (TfrX) and transcription fraction Y (TfrY), are required to fully reconstitute selective transcription of the VA I and tRNAAla2 DNA templates. TfrX contains one or more components required for forming transcriptional pre-emptive complexes, as shown in order of addition experiments. TfrX strongly protects a DNA segment surrounding the highly conserved distal sequence (the so-called B block) of the VA I and tRNAAla2 genes from DNase I digestion; we have also characterized weak protection of other segments of these genes by TfrX. DNase I protection experiments with TfrX and probes prepared from deletion variants of the VA I gene show that an intact B block is required for the strong protection.