Recent work has demonstrated a repressive effect of chromatin on the transcription of the yeast SNR6 gene in vitro. Here, we show the relations between chromatin structure and transcriptional activity of this gene in vivo. Analysis of the SNR6 locus by micrococcal nuclease digestion showed a protection of the TATA box, nuclease-sensitive sites around the A and B blocks, and arrays of positioned nucleosomes in the flanking regions. Analysis of a transcriptionally silent SNR6 mutant containing a 2-bp deletion in the B block showed a loss of TATA-protection and rearrangement or destabilization of nucleosomes in the flanking regions. Hence, SNR6 organizes the chromatin structure in the whole region in a manner dependent on its transcriptional state. Transcriptional analysis was performed by use of maxi-gene SNR6 constructs introduced into histone-mutated strains. Chromatin disruption induced by histone H4 depletion stimulated the transcription of promoter-deficient, but not of wild-type SNR6 genes, revealing a competition between the formation of nucleosomes and the assembly of Pol III transcription complexes that was much in favor of transcription factors. On the other hand, amino-terminal mutations in histone H3 or H4 had no effect (H4) or only a moderate stimulatory effect (H3) on the transcription of promoter-deficient SNR6 genes.
Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6A⌬ nhp6B⌬ double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6A⌬ nhp6B⌬ cells at 37°C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6A⌬ nhp6B⌬ cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxilliary factors are required for the optimal transcription of at least some specific Pol III genes.Transcription of small genes by RNA polymerase III (Pol III) in yeast involves a multistep assembly of transcription factors into a preinitiation complex which recruits RNA Pol III (for a review, see reference 35). The A and B blocks found in most Pol III promoters are first recognized by a multisubunit complex called Pol III transcription factor C (TFIIIC). TFIIIC, one of the largest and most complex transcription factors known, has a molecular mass of about 600 kDa and is composed of six subunits. It acts as an assembly factor to direct the binding of the initiation factor TFIIIB to an upstream gene position. Once assembled into a highly stable protein-DNA complex at Pol III promoters, TFIIIB can direct multiple rounds of transcription by Pol III in vitro in the absence of TFIIIC (17,18). TFIIIB is composed of three loosely associated polypeptides, the TATA-binding protein (19), a general transcription factor used by all eukaryotic and archeal RNA polymerases (14, 27); BЉ or TFIIIB90, which displays little homology to other known proteins except for a putative SANT domain (1,20,28,29); and Brf1 or TFIIIB70, which shows 44% similarity to TFIIB in its N-terminal 320 residues (3,7,21).In addition to these basal factors, there are hints that additional components exist which influence transcription efficiency or accuracy. A protein called TFIIIE, which has yet to be characterized, is able to stimulate transcription under certain conditions (9, 29). TFIIIE has been suggested to act by facilitating TFIIIB recruitment, by inducing conformational rearrangements of TFIIIB, or by stabilizing transcription complexes. A partially purified BЉ fraction was found to direct a more efficient and more accurate transcription initiation than the recombinant TFIIIB90 protein (6, 29), but the factors p...
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