Abstract
Transcriptional activators Staf and Oct-1 play critical roles in the activation of small nuclear RNA (snRNA) and snRNA-type gene transcription. Recently, we established that Staf binding to the human U6 snRNA (hU6) and Xenopus selenocysteine tRNA (xtRNA(Sec)) genes requires different sets of the seven C2-H2 zinc fingers. In this work, using a combination of oocyte microinjection, electrophoretic mobility shift assays, and missing nucleoside experiments with wild-type and mutant promoters, we demonstrate that the hU6 gene requires zinc fingers 2-7 for Staf binding and Oct-1 for maximal transcriptional activity. In contrast, the xtRNA(Sec) gene needs the binding of the seven Staf zinc fingers, but not Oct-1, for optimal transcriptional capacity. Mutation in the binding site for Staf zinc finger 1 in the tRNA(Sec) promoter reduced both Staf binding and transcriptional activity. Conversely, introduction of a zinc finger 1 binding site in the hU6 promoter increased Staf binding but interfered with the simultaneous Staf and Oct-1 binding, thus reducing transcriptional activity. Collectively, these results show that the differential utilization of Staf zinc finger 1 represents a new, critical determinant of the transcriptional activation mechanism for the Xenopus tRNA(Sec) and human U6 snRNA genes.
Highlights
Formation of a higher order transcription complex requires the interplay between specific DNA sequences and DNA-binding transcription factors
The GCG sequence located in the 3Ј part of xtRNASec distal sequence element (DSE) was changed to CAT, a sequence naturally found in the human U6 small nuclear RNAs (snRNAs) promoter (hU6) DSE, yielding mut GCG (Fig. 2A, construct 3)
The mutant hU6 template carrying the GCG sequence, exhibited a 2-fold reduction of template activity relative to the wt template (Fig. 2D, compare lanes 1 and 3). These results indicate that the presence of a GCG sequence at identical locations in tRNASec and U6 DSEs can lead to opposite effects on transcriptional activities, depending on the gene promoter context
Summary
Formation of a higher order transcription complex requires the interplay between specific DNA sequences and DNA-binding transcription factors. Substitution of the GCG sequence in the xtRNASec Staf element (mut GCG in Fig. 2A) resulted in a marked decrease in template activity to 40% of the wild-type level (Fig. 2C, compare lanes 1 and 3). These results indicate that the presence of a GCG sequence at identical locations in tRNASec and U6 DSEs can lead to opposite effects on transcriptional activities, depending on the gene promoter context.
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