The differential expression of the Xenopus laevis somatic and oocyte 5S RNA genes is partially, but not solely, due to several base differences near the 5' boundary of the internal control region. A hybrid oocyte 5S gene with somatic-type base changes at +47, +53, +55, and +56 had intermediate transcriptional activity in oocyte S150 extracts. These base substitutions also resulted in increased affinity for a factor(s), other than TFIIIA, which forms a stable complex with the 5S gene.
The differential expression of the Xenopus laevis somatic and oocyte 5S RNA genes is partially, but not solely, due to several base differences near the 5' boundary of the internal control region. A hybrid oocyte 5S gene with somatic-type base changes at +47, +53, +55, and +56 had intermediate transcriptional activity in oocyte S150 extracts. These base substitutions also resulted in increased affinity for a factor(s), other than TFIIIA, which forms a stable complex with the 5S gene.
DNase I footprints and affinity measurements showed that the C-terminal arm of Xenopus transcription factor IIIA interacts differently with different Xenopus 5S DNAs, forming three distinct types of transcription factor IIIA-5S DNA complexes: a somatic type, a major-oocyte (and pseudogene) type, and a trace-oocyte type. Site-directed mutagenesis on the major-oocyte 5S gene revealed that somatic-type changes at positions 53, 55, and 56 changed the structure of the transcription factor IIIA-5S DNA complex from major-oocyte to somatic, and a single trace-oocyte change at position 56 caused the change from major-oocyte to trace-oocyte complex. We further show that the somatic-type changes are accompanied by a marked enhancement in the rate of 5S RNA transcription, and we discuss the possible biological relevance of these findings.
DNase I footprints and affinity measurements showed that the C-terminal arm of Xenopus transcription factor IIIA interacts differently with different Xenopus 5S DNAs, forming three distinct types of transcription factor IIIA-5S DNA complexes: a somatic type, a major-oocyte (and pseudogene) type, and a trace-oocyte type. Site-directed mutagenesis on the major-oocyte 5S gene revealed that somatic-type changes at positions 53, 55, and 56 changed the structure of the transcription factor IIIA-5S DNA complex from major-oocyte to somatic, and a single trace-oocyte change at position 56 caused the change from major-oocyte to trace-oocyte complex. We further show that the somatic-type changes are accompanied by a marked enhancement in the rate of 5S RNA transcription, and we discuss the possible biological relevance of these findings.
The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level 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 TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation 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 from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.
Effect of sequence differences between somatic and oocyte 5S RNA genes on transcriptional efficiency in an oocyte S150 extract.