TFIIIB placement on a yeast U6 RNA gene in vivo is directed primarily by TFIIIC rather than by sequence-specific DNA contacts.
The Saccharomyces cerevisiae U6 RNA gene (SNR6), which is transcribed by RNA polymerase III, has an unusual combination of promoter elements: an upstream TATA box, an intragenic A block, and a downstream B block. In tRNA genes, the A and B blocks are binding sites for the transcription initiation factor TFIIIC, which positions TFIIIB a fixed distance upstream of the A block. However, in vitro transcription of SNR6 with purified components requires neither TFIIIC nor the A and B blocks, presumably because TFIIIB recognizes the upstream sequences directly. Here we demonstrate that TFIIIB placement on SNR6 in vivo is directed primarily by the TFIIIC-binding elements rather than by upstream sequences. We show that the A block is a stronger start site determinant than the upstream sequences when the two are uncoupled by an insertion mutation. Furthermore, while TFIIIC-independent in vitro transcription of SNR6 is highly sensitive to TATA box point mutations, in vivo initiation on SNR6 is only marginally sensitive to such mutations unless the A block is mutated. Intriguingly, a deletion downstream of the U6 RNA coding region that reduces A-to-B block spacing also increases in vivo dependence on the TATA box. Moreover, this deletion results in the appearance of micrococcal nuclease-hypersensitive sites in the TFIIIB chromatin footprint, indicating that TFIIIB binding is disrupted by a mutation 150 bp distant. This and additional chromatin footprinting data suggest that SNR6 is assembled into a nucleoprotein complex that facilitates the TFIIIC-dependent binding of TFIIIB.
Gerlach
V L
VL
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706-1532.
Whitehall
S K
SK
Geiduschek
E P
EP
Brow
D A
DA
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D016385:TATA Box; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
D017931:DNA Primers; D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC