▪ Abstract The events leading to transcription of eukaryotic protein-coding genes culminate in the positioning of RNA polymerase II at the correct initiation site. The core promoter, which can extend ∼35 bp upstream and/or downstream of this site, plays a central role in regulating initiation. Specific DNA elements within the core promoter bind the factors that nucleate the assembly of a functional preinitiation complex and integrate stimulatory and repressive signals from factors bound at distal sites. Although core promoter structure was originally thought to be invariant, a remarkable degree of diversity has become apparent. This article reviews the structural and functional diversity of the RNA polymerase II core promoter.

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The RNA polymerase II core promoter.

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Δ strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Δ mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Transcriptional Profiling Shows that Gcn4p Is a Master Regulator of Gene Expression during Amino Acid Starvation in Yeast

http://www.alliot.fr/BIO/PDF/nihms-17486.pdf

Prevalence of the initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters.

Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.

The ACT domain family.

The crystal structure of an allosteric chorismate mutase, the Thr-226-->Ile mutant, from yeast Saccharomyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method. Solvent-flattening and electron-density modification were applied for phase improvement. The current crystallographic R factor is 0.196. The final model includes 504 of the 512 residues and 97 water molecules. In addition, two tryptophan molecules were identified in the interface between monomers. The overall structure is completely different from the reported structure of chorismate mutase from Bacillus subtilis. This structure showed 71% helices with essentially no beta-sheet structures.

https://www.pnas.org/content/pnas/91/23/10814.full.pdf

The crystal structure of allosteric chorismate mutase at 2.2-A resolution

The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.

Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae.

The yeast TRP4 promoter contains three responsive elements (GCREs) for the 'general control' transcriptional activator GCN4, which are arranged in two upstream elements, UAS1 (GCRE1) and UAS2 (GCRE2 and GCRE3). A point mutation analysis of these elements revealed that all three GCREs are required for GCN4-dependent transcription, but none are involved in basal transcription. Basal transcription and GCN4-dependent transcription use completely different initiator elements in the TRP4 promoter. UAS1 acts synergistically with UAS2 to activate the GCN4-dependent transcription of TRP4. A consensus TATA box can functionally replace the UAS2 element to allow normal GCN4-dependent transcription, suggesting that UAS2 is analogous to the TATA element of other promoters. GCN4 might therefore activate transcription by exhibiting two alternative functions within the natural TRP4 promoter.

Three GCN4 responsive elements act synergistically as upstream and as TATA-like elements in the yeast TRP4 promoter.

The gene AR07 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae. We investigated the transcription of the AR07 gene. Three 5′ ends at positions − 36, − 56 and − 73 and the 3′ end of the transcripts 146 bp downstream of the translational stop codon were mapped. As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point. This element binds GCN4 specifically in vitro. Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo. We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.

A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae.

We have cloned and sequenced a Saccharomyces cerevisiae gene coding for a protein with significant similarities to the mitochondrial carrier family. The gene we termed YMC1 (yeast mitochondrial carrier) is located on chromosome XVI, closely downstream of ARO7 encoding chorismate mutase.

Roney Graf

Papers

The crystal structure of allosteric chorismate mutase at 2.2-A resolution

Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae.

Three GCN4 responsive elements act synergistically as upstream and as TATA-like elements in the yeast TRP4 promoter.

A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae.

YMC1, a yeast gene encoding a new putative mitochondrial carrier protein.