No abstract

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8-and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5A, ada2A, or ada3A yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5A and spt7A strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (_Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).[Key Words." Acetyltransferase; nucleosome; transcription; Spt; Ada; Gcn5] Received March 28, 1997; revised version accepted May 15, 1997.Chromatin structure has an intricate role in the regulation of eukaryotic gene transcription. Nucleosomes suppress basal transcription initiation in vivo and in vitro increasing the dependence of transcription on the function of sequence-specific activator proteins (for review, see Grunstein 1990; Owen-Hughes and Workman 1994). Chromatin structures are remodeled before or during transcription activation generating DNase I hypersensitive regions (DHSs) at transcription control elements (Hager et al. 1995;Steger and Workman 1996;Svaren and Horz 1996). Multiprotein complexes have been impliSCorresponding authors.

We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Transcription by RNA polymerase II in Saccharomyces cerevisiae and in humans is widespread, even in genomic regions that do not encode proteins. The purpose of such intergenic transcription is largely unknown, although it can be regulatory. We have discovered a role for one case of intergenic transcription by studying the S. cerevisiae SER3 gene. Our previous results demonstrated that transcription of SER3 is tightly repressed during growth in rich medium. We now show that the regulatory region of this gene is highly transcribed under these conditions and produces a non-protein-coding RNA (SRG1). Expression of the SRG1 RNA is required for repression of SER3. Additional experiments have demonstrated that repression occurs by a transcription-interference mechanism in which SRG1 transcription across the SER3 promoter interferes with the binding of activators. This work identifies a previously unknown class of transcriptional regulatory genes.