Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.     

Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene

We show that the yeast HAP1 activator locus encodes a protein that binds in vitro to the upstream activation site, UAS1, of the CYC1 gene (iso-1-cytochrome c). Binding of wild-type HAP1 and truncated HAP1 derivatives to UAS1 is evident in crudely fractionated yeast extracts using the gel electrophoresis DNA binding assay. The binding of HAP1 in vitro, like the activity of UAS1 in vivo, is stimulated by heme. HAP1 binds to region B, one of two portions of UAS1 shown to be important by genetic analysis of the site. Surprisingly, HAP1 binds to the same sequence as a second factor, RC2. Both HAP1 and RC2 bind to the same side of the helix, and make similar but not identical major and minor groove contacts that span two full turns. An additional factor that binds to the second important part of UAS1, the region A factor (RAF), is also identified. A model depicting the interplay of HAP1, RC2, and RAF in the control of UAS1 is presented.     

酵母SUC2基因上游顺序对其表达的影响

从SUC2基因上游约—900bp向起始密码进行系列缺失。将带有这种缺失上游区的SUC2基因插入多拷贝质粒,并转化进不产蔗糖酶的酵母细胞。测定了这些缺失株表达蔗糖酶的数量。结果表明:在葡萄糖阻遏条件下,SUC2上游区缺失从-636bp到-179bp的不同细胞,糖基化蔗糖酶的表达量逐渐升高。和野生型相比,SUC2上游区缺失到-223bp和-179bp的细胞糖基化蔗糖酶量增加100倍以上。在葡萄糖去阻遏条件下,SUC2上游缺失从-395bp到-179bp的不同细胞,糖基化蔗糖酶的表达量只显示微弱的去阻遏效应。缺失末端达-89bp和-41bp的细胞只表达很少的糖基化蔗糖酶,但是非糖基化蔗糖酶的表达量明显增加。     

Yeast HAP1 activator binds to two upstream activation sites of different sequence

We show that the HAP1 protein binds in vitro to the upstream activation site (UAS) of the yeast CYC7 gene. Strikingly, this sequence bears no obvious similarity to the sequence bound by HAP1 at UAS1 of the CYC1 gene. The CYC1 and CYC7 sites compete for binding to HAP1 and have comparable affinities for the protein. The gross features of the interaction of HAP1 with the two sites are similar: multiple major and minor groove contacts, spanning 23 bp, on one helical face, with a back-side major groove contact toward one end. The precise positions of the contacts differ, however. A mutant form of HAP1, HAP1-18, abolishes the ability of the protein to bind to UAS1 but not CYC7 DNA. Possible mechanisms for how a single protein recognizes two sequences are discussed.