Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.

Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.

Nrg1 functions as a global transcriptional repressor of glucose-repressed genes through its direct binding to the specific promoter regions.

Nrg1 is a zinc finger protein involved in the glucose repression of several glucose-repressed genes such as STA1, SUC2, and GAL1. Although the molecular details of the Nrg1-mediated repression of STA1 have been partly characterized, it still remains largely unknown how Nrg1 regulates these multiple target genes. In this study, we show that Nrg1 mediates the glucose repression of SUC2 and HXT2 through its direct binding to the specific promoter regions; it binds to the -404 to -360 region of the SUC2 promoter and the -957 to -810 region of the HXT2 promoter. Nrg1 also interacts with the -380 to -250 region of the PCK1 promoter, suggesting that it might also contribute to the PCK1 repression. In addition, ChIP assays confirmed that Nrg1 associated with specific promoter regions of these glucose-repressed genes in vivo. Analysis of the DNA fragments to which it binds indicates that Nrg1 may recognize T/ACCCC sequence within the promoters of these glucose-repressed genes as well as in its own promoter. Collectively, our findings indicate that Nrg1 mediates the glucose repression of multiple genes through its direct binding to the specific promoter regions.

The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4.

We report the complete genome sequence of Zymomonas mobilis ZM4 (ATCC31821), an ethanologenic microorganism of interest for the production of fuel ethanol. The genome consists of 2,056,416 base pairs forming a circular chromosome with 1,998 open reading frames (ORFs) and three ribosomal RNA transcription units. The genome lacks recognizable genes for 6-phosphofructokinase, an essential enzyme in the Embden-Meyerhof-Parnas pathway, and for two enzymes in the tricarboxylic acid cycle, the 2-oxoglutarate dehydrogenase complex and malate dehydrogenase, so glucose can be metabolized only by the Entner-Doudoroff pathway. Whole genome microarrays were used for genomic comparisons with the Z. mobilis type strain ZM1 (ATCC10988) revealing that 54 ORFs predicted to encode for transport and secretory proteins, transcriptional regulators and oxidoreductase in the ZM4 strain were absent from ZM1. Most of these ORFs were also found to be actively transcribed in association with ethanol production by ZM4.

Recruitment of the Swi/Snf complex by Ste12-Tec1 promotes Flo8-Mss11-mediated activation of STA1 expression.

In the yeast Saccharomyces diastaticus, expression of the STA1 gene, which encodes an extracellular glucoamylase, is activated by the specific DNA-binding activators Flo8, Mss11, Ste12, and Tec1 and the Swi/Snf chromatin-remodeling complex. Here we show that Flo8 interacts physically and functionally with Mss11. Flo8 and Mss11 bind cooperatively to the inverted repeat sequence TTTGC-n-GCAAA (n = 97) in UAS1-2 of the STA1 promoter. In addition, Flo8 and Mss11 bind indirectly to UAS2-1 of the STA1 promoter by interacting with Ste12 and Tec1, which bind to the filamentation and invasion response element (FRE) in UAS2-1. Furthermore, our findings indicate that the Ste12, Tec1, Flo8, and Mss11 activators and the Swi/Snf complex bind sequentially to the STA1 promoter, as follows: Ste12 and Tec1 bind first to the FRE, whereby they recruit the Swi/Snf complex to the STA1 promoter. Next, the Swi/Snf complex enhances Flo8 and Mss11 binding to UAS1-2. In the final step, Flo8 and Mss11 directly promote association of RNA polymerase II with the STA1 promoter to activate STA1 expression. In the absence of glucose, the levels of Flo8 and Tec1 are greatly increased, whereas the abundances of two repressors, Nrg1 and Sfl1, are reduced, suggesting that the balance of transcriptional regulators may be important for determining activation or repression of STA1 expression.

Glucose repression of STA1 expression is mediated by the Nrg1 and Sfl1 repressors and the Srb8-11 complex.

In the yeast Saccharomyces diastaticus, expression of the STA1 gene, which encodes an extracellular glucoamylase, is negatively regulated by glucose. Here we demonstrate that glucose-dependent repression of STA1 expression is imposed by both Sfl1 and Nrg1, which serve as direct transcriptional repressors. We show that Nrg1 acts only on UAS1, and Sfl1 acts only on UAS2, in the STA1 promoter. When bound to its specific site, Sfl1 (but not Nrg1) prevents the binding to UAS2 of two transcriptional activators, Ste12 and Tec1, required for STA1 expression. We also found that Sfl1 contributes to STA1 repression by binding to the promoter and inhibiting the expression of FLO8, a gene that encodes a third transcriptional activator involved in STA1 expression. In addition, we show that the levels of Nrg1 and Sfl1 increase in glucose-grown cells, suggesting that the effects of glucose are mediated, at least in part, through an increase in the abundance of these repressors. NRG1 and SFL1 expression requires the Srb8-11 complex, and correspondingly, the Srb8-11 complex is also necessary for STA1 repression. However, our evidence indicates that the Srb8-11 complex does not associate with either the SFL1 or the NRG1 promoter and thus plays an indirect role in activating NRG1 and SFL1 expression.

Regulation of penicillin G acylase gene expression in Escherichia coli by repressor PaaX and the cAMP-cAMP receptor protein complex.

The pga gene of Escherichia coli W ATCC11105 encodes a penicillin G acylase whose expression is regulated at both the transcriptional and post-transcriptional level. In this work we have shown that PaaX is the repressor of pga expression, and we have identified its binding consensus as TGATTC(N27)GAATCA. We conclude that the process of "PAA induction" actually involves relief of pga from repression by PaaX. Other features of the pga promoter have also been characterized. (i) It has a native class III cAMP-receptor protein (CRP)-dependent promoter with two CRP-binding sites. (ii) The downstream CRP-binding site II has higher affinity. (iii) Binding of cAMP-CRP to both sites (I + II) is required for maximal expression. We have also shown that the PaaX-binding site overlaps with the CRP-binding site I. This implies that PaaX and the cAMP-CRP compete for binding to the region around the CRP-binding site I and therefore have antagonistic effects on pga expression.

STA10 repression of STA gene expression is caused by a defective activator, flo8, in Saccharomyces cerevisiae.

The expression of STA genes that encode extracellular glucoamylase isozymes is repressed in most laboratory Saccharomyces cerevisiae strains, which are believed to contain an undefined repressor, designated STA10. To identify the regulator involved in STA10 repression, we investigate the FLO8, MSN1, MSS11, STE12, and TEC1 genes. The Deltaflo8 or Deltamss11 deletion mutants in the sta10 genetic background exhibit both a loss of flocculation ability and a reduction in extracellular glucoamylase activity, as in the STA10 strain. Moreover, the STA10 repression is suppressed completely or partially by the introduction of a single copy of the FLO8 or MSS11 genes. Sequence analysis and complementation testing of the STA10 strain reveal that it has an inactive, mutated flo8-1 allele. A random spore analysis and transplacement (allele replacement) experiment confirms that the repressive phenotype of STA10 is due to the amber mutation of the transcriptional activator, FLO8.

Multiple developmental defects derived from impaired recruitment of ASC-2 to nuclear receptors in mice: implication for posterior lenticonus with cataract.

ASC-2, a recently isolated transcriptional coactivator molecule, stimulates transactivation by multiple transcription factors, including nuclear receptors. We generated a potent dominant negative fragment of ASC-2, encompassing the N-terminal LXXLL motif that binds a broad range of nuclear receptors. This fragment, termed DN1, specifically inhibited endogenous ASC-2 from binding these receptors in vivo, whereas DN1/m, in which the LXXLL motif was mutated to LXXAA to abolish the receptor interactions, was inert. Interestingly, DN1 transgenic mice but not DN1/m transgenic mice exhibited severe microphthalmia and posterior lenticonus with cataract as well as a variety of pathophysiological phenotypes in many other organs. Our results provide a novel insight into the molecular and histopathological mechanism of posterior lenticonus with cataract and attest to the importance of ASC-2 as a pivotal transcriptional coactivator of nuclear receptors in vivo.