酿酒酵母非发酵代谢调控
2007-06-17 14:48:04   来源：网络数据库   评论：0 点击：

sed expression of the iso-2-cytochrome c gene CYC7 (Rothstein and Sherman 1980), by screening for constitutive invertase activity(Trumbly 1986) and as suppressors of snf1 (Carlson et al. 1984). Overexpression of CYC8 reduced the activity of invertase, even under derepression conditions (Schultz and Carlson 1987). The CYC8 gene product is a nuclear phosphoprotein and contains ten tetratricopeptide repeats (TPR; Sikorski et al. 1990) which are essential for protein function (Schultz et al. 1990). TPR motifs 1–3 of Cyc8 are required for interaction with a second pleiotropic repressor, Tup1 (Tzamarias and Struhl 1995), which is also known as Flk1, Cyc9, Umr7,Rox4, Slf2, Aar1, Aer2 or Amm1. These alternative designations indicate the multitude of tup1 mutant phenotypes which are quite similar to those of cyc8 mutants, arguing for a common molecular function of Cyc8 and Tup1. Tup1 contains six (possibly seven) C-terminal WD-40 repeats (b-transducin motif; Williams and Trumbly 1990; Van der Voorn and Ploegh 1992), presumably mediating multiple protein–protein interactions. Regions of Tup1 distinct from the WD-40 repeats are absolutely required for interaction with Cyc8 and for the onset of target-gene repression (Tzamarias and Struhl 1994). It has been proposed that the Cyc8-Tup1 complex (molecular stoichiometry is one subunit of Cyc8 with four subunits of Tup1; Varanasi et al.1996) does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA-binding proteins (Keleher et al. 1992; reviewed by Smith and Johnson 2000). The specificity factor required for glucose-repressible genes is Mig1. As outlined above, the C-terminal repression effector domain of Mig1 binds to Cyc8. Transcriptional repression by Cyc8-Tup1 has been also shown with the purified complex in vitro (Redd et al. 1997). When artificially tethered to a test promoter, Tup1 is able to repress transcription, even in the absence of Cyc8 (Tzamarias and Struhl 1994). Thus, Cyc8 may serve as a link between the genuine repressor Tup1 and the pathway-specific DNA-binding proteins.

Tup1-dependent gene repression may utilize at least two mechanisms. First, Tup1 influences the organization of chromatin by direct contact of its repression domain with the N-termini of histones H3 and H4 (Edmondson et al. 1996). Since these interactions are substantially weakened when histone tails are acetylated, promoters repressed by Tup1 are associated with underacetylated histones in vivo (Bone and Roth 2001; Deckert and Struhl 2001). Local deacetylation of histones may be the result of direct interaction of histone deacetylases Rpd3 and Hos2 with Cyc8-Tup1. Consequently, histone hyperacetylation caused by combined mutations in the histone deacetylase genes RPD3, HOS1 and HOS2 abolishes repression by Cyc8-Tup1 (Watson et al. 2000). Tup1 also recruits the H3/H2B-specific deacetylase Hda1 (Wu et al. 2001), arguing for some redundancy in histone modification occurring under repressing conditions. Second, Tup1 negatively affects the general transcriptional machinery by interacting with subunits of the Srb/mediator complex of the RNA polymerase II holoenzyme. Repression by Cyc8-Tup1 was less effective with mutant alleles of SRB7 (Gromo¨ ller and Lehming 2000), SRB8, SRB9, SRB10, SRB11 (Kuchin and Carlson 1998; Lee et al. 2000) and MED3 (Papamichos-Chronakis et al. 2000). At least for Srb7 and Med3, direct contact with Cyc8-Tup1 could be demonstrated. In conclusion, Cyc8-Tup1 may counteract transcription by its negative influence on the Srb/mediator complex, possibly by preventing holoenzyme recruitment to the basal promoter.

In addition to the repressor function of Cyc8-Tup1, growing evidence for a positive role accumulates. Initially, a positive effect of Cyc8-Tup1 on Hap1-dependent gene expression was shown (Zhang and Guarente 1994). More recent work described the interaction of Cyc8-Tup1 with the activation domain of Rtg3, a transcription factor binding to the control region of the citrate synthase gene, CIT2 (Conlan et al. 1999; see below). Even for the GAL1 promoter, which is clearly repressed by Cyc8-Tup1 in the presence of glucose, a positive function could be demonstrated under conditions of galactose induction. In contrast to previous hypotheses, Cyc8-Tup1 remain bound at the promoter and, together with the mediator protein Cti6, contribute to recruitment of the SAGA coactivator complex (Papamichos-Chronakis et al. 2002). These results are consistent with our data showing a substantially reduced expression of gluconeogenic genes in cyc8 and tup1 null mutants (to about 30% of the wild-type level; S. Roth and H.-J. Schu¨ ller, unpublished data).

Mutants insensitive to glucose repression were also isolated by selection for resistance against 2-deoxyglucose (Zimmermann and Scheel 1977). The regulatory defect of hxk2 (hex1), reg1 (hex2), grr1 (cat80) and glc7 (cid1, dis2) mutants (Entian and Zi