Analysis of conditional mutants in selected RNA polymerase subunits
revealed that two others, in addition to the RPB1 carboxy terminal
domain (CTD), are essential for normal transcriptional activation. In
vitro transcription with extracts prepared from mutants in either of
these two subunits, RPB3 or RPB5, revealed defects in activation by
GAL4-VP16. These data are corroborated in vivo at several promoters
using inducible reporter plasmids and Northern analysis. Consistent with
its abnormal activation phenotype, the RPB5 point mutant maps to a
conserved region of its human counterpart implicated by others to play a
role in activation of human genes. In fact, a human-yeast RPB5 chimera
containing this species-specific activation region can now support yeast
cell growth. Cells with activation mutations in both RPB5 and the RPB1-CTD do not display additive or synergistic impairment of activation. The
RPB3 activation mutant contains two point mutations, one in a highly
conserved, eukaryotic-specific, zinc-finger and another in a region
corresponding to the recently defined activation target in the N-terminal domain of the alpha subunit of bacterial RNA polymerase.
Characterization of either RPB3 point mutation alone revealed discrete,
non-overlapping activation defects that were less extreme than those of
the severely crippled double mutant. Our RPB3 data solidifies its
functional relationship with alpha and uncovers new activation target
sites missing in alpha.
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