ROLE OF TFIIIA ZINC FINGERS IN-VIVO - ANALYSIS OF SINGLE-FINGER FUNCTION IN DEVELOPING XENOPUS-EMBRYOS

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) ha s nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc finger s to the activity of TFIIIA in living cells. Nine separate TFIIIA muta nts were expressed in xenopus embryos following microinjection of thei r respective in vitro-derived mRNAs. Each mutant contained a single hi stidine-to-asparagine substitution in the third zinc ligand position o f an individual zinc finger. These mutations result in structural disr uption of the mutated finger with little or no effect on the other fin gers. The activity of mutant proteins in vivo was assessed by measurin g transcriptional activation of the endogenous 5S RNA genes. Mutants c ontaining a substitution in zinc finger 1, 2, or 3 activate 5S RNA gen es at a level which is reduced relative to that in embryos injected wi th the message for wild-type TFIIIA. Proteins with a histidine-to-aspa ragine substitution in zinc finger 5 or 7 activate 5S RNA genes at a l evel that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFII IA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutatio n in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result f rom differences in the relative number and/or activity of transcriptio n complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcript ional activation of the normally inactive oocyte-type 5S RNA genes. Th e remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Di sruption of zinc finger 6 results in a form of TFIIIA that exhibits re duced susceptibility to feedback inhibition by 5S RNA and therefore in creases the availability of the transcription factor for transcription complex formation.