Upstream activating sequence

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An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the enhancer in multicellular eukaryotes. [1] Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. [2] The upstream activation sequence is found adjacently upstream to a minimal promoter (TATA box) and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin. [3] To further understand the function of an upstream activation sequence, it is beneficial to see its role in the cascade of events that lead to transcription activation. The pathway begins when activators bind to their target at the UAS recruiting a mediator. A TATA-binding protein subunit of a transcription factor then binds to the TATA box, recruiting additional transcription factors. The mediator then recruits RNA polymerase II to the pre-initiation complex. Once initiated, RNA polymerase II is released from the complex and transcription begins. [4]

Contents

Examples

GAL1-GAL10 intergenic region (UASG)

The property of the GAL1-GAL10 to bind the GAL4 protein is utilised in the GAL4/UAS technique for controlled gene mis-expression in Drosophila. This is the most popular form of binary expression in Drosophila melanogaster, a system which has been adapted for many uses to make Drosophila melanogaster one of the most genetically tractable multicellular organisms. [5] In this technique, four related binding sites between the GAL10 and GAL1 loci in Saccharomyces cerevisiae serve as an Upstream Activating Sequences (UAS) element through GAL4 binding. [6] Several studies have been conducted with Saccharomyces cerevisiae to explore the exact function of upstream activation sequences, often focusing on the aforementioned GAL1-GAL10 intergenic region. [7] The consensus is 5′-CGG-N11-CCG-3′. [8]

One study explored the galactose-responsive upstream activation sequence (UASG), looking at the influence of proximity to this UAS for nucleosome positioning. Proximity to the UAS was chosen because deletions of DNA flanking the UAS left the nucleosome array unaltered, indicating that nucleosome positioning was not related to sequence-specific histone-DNA interactions. The role of specific regions of UASG was analyzed by inserting oligonucleotides with different binding properties, leading to the successful identification of a region responsible for the creation of an ordered array. The sequence identified overlapped a binding site for GAL4 protein, which is a positive regulator for transcription which coincides with the function of upstream activating sequences. [9]

Another study looked at the effect of inserting the UASG into the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) . This hybrid promoter was then utilized to express human immune interferon, a toxic substance to yeast that results in a reduced copy number and low plasmid stability. Relative to the native promoter, expression of the hybrid promoter was induced roughly 150- to 200-fold in the cultures by growth in galactose, induction that wasn't apparent with glucose as the carbon source. When compared to the native GPD promoter, the presence of UASG caused the transcriptional activity to remain equivalently enhanced under induced conditions. [10]

Inositol-sensitive upstream activation sequence (UASINO)

The inositol-sensitive upstream activation sequence (UASINO) has a consensus sequence 5'-CATGTGAAAT-3' and is present in the promoter regions of genes that encode enzymes of phospholipid biosynthesis. These enzymes are regulated by inositol and choline, both of which are phospholipid precursors. Within this consensus sequence, the first six bases are homologous with canonical binding motif for proteins within the bHLH or the basic helix-loop-helix family. Studies have shown that Ino2p and Ino4p, two bHLH regulatory proteins from Saccharomyces cerevisiae, bind to promoter fragments containing this element of the consensus sequence. Additional studies have been designed to explore the function of UASINO in more detail largely in part because a large number of phospholipid biosynthetic enzyme activities in the model organism Saccharomyces cerevisiae show this common pattern of expression. [11]

One study explored the interaction between Ino4p and Ino2p in more depth, examining the dimerization that takes place between the two prior to binding to the promoter of the INO1 gene and activating transcription. By isolating 31 recessive suppressors of the ino4-8 mutant of yeast and determining that 29 were of the same locus, the researchers identified the locus as REG1 . One allele of REG1, the suppressor mutant sia1-1, was capable of suppressing the inositol auxotrophy, revealing a possible pathway for the repression of inositol-sensitive upstream activating sequence-containing genes of yeast. [12]

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<span class="mw-page-title-main">Nucleosome</span> Basic structural unit of DNA packaging in eukaryotes

A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4.

In molecular biology, the TATA box is a sequence of DNA found in the core promoter region of genes in archaea and eukaryotes. The bacterial homolog of the TATA box is called the Pribnow box which has a shorter consensus sequence.

<span class="mw-page-title-main">Regulation of gene expression</span> Modifying mechanisms used by cells to increase or decrease the production of specific gene products

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.

RNA polymerase 1 is, in higher eukaryotes, the polymerase that only transcribes ribosomal RNA, a type of RNA that accounts for over 50% of the total RNA synthesized in a cell.

<span class="mw-page-title-main">Two-hybrid screening</span> Molecular biology technique

Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.

An insulator is a type of cis-regulatory element known as a long-range regulatory element. Found in multicellular eukaryotes and working over distances from the promoter element of the target gene, an insulator is typically 300 bp to 2000 bp in length. Insulators contain clustered binding sites for sequence specific DNA-binding proteins and mediate intra- and inter-chromosomal interactions.

The Ty5 is a type of retrotransposon native to the Saccharomyces cerevisiae organism.

<span class="mw-page-title-main">GAL4/UAS system</span> Biochemical method

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It is based on the finding by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988 that Gal4 binding to UAS sequences activates gene expression. The method was introduced into flies by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS, an enhancer to which GAL4 specifically binds to activate gene transcription.

<span class="mw-page-title-main">Inositol-3-phosphate synthase</span>

In enzymology, an inositol-3-phosphate synthase is an enzyme that catalyzes the chemical reaction

The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. Repression of gene expression for this operon works via binding of repressor molecules to two operators. These repressors dimerize, creating a loop in the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus prevent transcription. Additionally, since the metabolism of galactose in the cell is involved in both anabolic and catabolic pathways, a novel regulatory system using two promoters for differential repression has been identified and characterized within the context of the gal operon.

G1/S-specific cyclin Cln3 is a protein that is encoded by the CLN3 gene. The Cln3 protein is a budding yeast G1 cyclin that controls the timing of Start, the point of commitment to a mitotic cell cycle. It is an upstream regulator of the other G1 cyclins, and it is thought to be the key regulator linking cell growth to cell cycle progression. It is a 65 kD, unstable protein; like other cyclins, it functions by binding and activating cyclin-dependent kinase (CDK).

<span class="mw-page-title-main">Pho4</span> Protein-coding gene in the species Saccharomyces cerevisiae S288c

Pho4 is a protein with a basic helix-loop-helix (bHLH) transcription factor. It is found in S. cerevisiae and other yeasts. It functions as a transcription factor to regulate phosphate responsive genes located in yeast cells. The Pho4 protein homodimer is able to do this by binding to DNA sequences containing the bHLH binding site 5'-CACGTG-3'. This sequence is found in the promoters of genes up-regulated in response to phosphate availability such as the PHO5 gene.

Cryptic unstable transcripts (CUTs) are a subset of non-coding RNAs (ncRNAs) that are produced from intergenic and intragenic regions. CUTs were first observed in S. cerevisiae yeast models and are found in most eukaryotes. Some basic characteristics of CUTs include a length of around 200–800 base pairs, a 5' cap, poly-adenylated tail, and rapid degradation due to the combined activity of poly-adenylating polymerases and exosome complexes. CUT transcription occurs through RNA Polymerase II and initiates from nucleosome-depleted regions, often in an antisense orientation. To date, CUTs have a relatively uncharacterized function but have been implicated in a number of putative gene regulation and silencing pathways. Thousands of loci leading to the generation of CUTs have been described in the yeast genome. Additionally, stable uncharacterized transcripts, or SUTs, have also been detected in cells and bear many similarities to CUTs but are not degraded through the same pathways.

SilentInformationRegulator (SIR) proteins are involved in regulating gene expression. SIR proteins organize heterochromatin near telomeres, ribosomal DNA (rDNA), and at silent loci including hidden mating type loci in yeast. The SIR family of genes encodes catalytic and non-catalytic proteins that are involved in de-acetylation of histone tails and the subsequent condensation of chromatin around a SIR protein scaffold. Some SIR family members are conserved from yeast to humans.

Gene gating is a phenomenon by which transcriptionally active genes are brought next to nuclear pore complexes (NPCs) so that nascent transcripts can quickly form mature mRNA associated with export factors. Gene gating was first hypothesised by Günter Blobel in 1985. It has been shown to occur in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster as well as mammalian model systems.

The transactivation domain or trans-activating domain (TAD) is a transcription factor scaffold domain which contains binding sites for other proteins such as transcription coregulators. These binding sites are frequently referred to as activation functions (AFs). TADs are named after their amino acid composition. These amino acids are either essential for the activity or simply the most abundant in the TAD. Transactivation by the Gal4 transcription factor is mediated by acidic amino acids, whereas hydrophobic residues in Gcn4 play a similar role. Hence, the TADs in Gal4 and Gcn4 are referred to as acidic or hydrophobic, respectively.

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<span class="mw-page-title-main">Pdr1p</span>

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<span class="mw-page-title-main">SPT20</span> Transcription factor

Transcription factor SPT20 is a regulator of transcription. It can recruit TATA binding protein (TBP) and possible other base factors to bind to TATA box. The model of its action by example Saccharomyces cerevisiae was studied. It functions as a component of the transcriptional regulatory complex histone-acetylation a (HAT) SAGA, SALSA and FIG. SAGA is involved in the regulation of transcription-dependent RNA polymerase II about 10% of the yeast gene. In promoter, SAGA is required to engage basal transcription mechanisms. Affects RNA polymerase II transcription activity through various activities, such as TBP interaction and promoter selectivity, interaction with transcription activators and modification chromatin by histone acetylation (GCN5) and ubiquitin deacetylation (UBP8). SAGA acetylates nucleosome or histone H3 to some extent.

References

  1. ↑ Webster, Nocholas; Jin, Jia Rui; Green, Stephen; Hollis, Melvyn; Chambon, Pierre (29 January 1988). "The Yeast UASG is a transcriptional enhancer in human hela cells in the presence of the GAL4 trans-activator". Cell. 52 (2): 169–178. doi:10.1016/0092-8674(88)90505-3. PMID   2830022. S2CID   26819676.
  2. ↑ West, Jr., Robert W.; Yocum, R. Rogers; Ptashne, Mark (November 1984). "Saccharomyces cerevisiae GAL1-GAL10 Divergenet Promoter Region: Location and Function of the Upstream Activating Sequence UASG". Molecular and Cellular Biology. 4 (11): 2467–2478. doi:10.1128/MCB.4.11.2467. PMC   369078 . PMID   6392852.
  3. ↑ Lewandoski, Mark (October 2001). "Conditional control of gene expression in the mouse". Nature Reviews Genetics. 2 (10): 743–755. doi:10.1038/35093537. PMID   11584291. S2CID   27099914.
  4. ↑ Wion, Didier; Casadesus, Josep (March 2006). "N6-methyl-adenine: An epigenetic signal for DNA-protein interactions". Nature Reviews Microbiology. 4 (3): 183–192. doi:10.1038/nrmicro1350. PMC   2755769 . PMID   16489347.
  5. ↑ Wimmer, Ernst A. (March 2003). "Applications of insect transgenesis". Nature Reviews Genetics. 4 (3): 225–232. doi:10.1038/nrg1021. PMID   12610527. S2CID   7668484.
  6. ↑ Duffy, Joseph B. (2002). "GAL4 system in Drosophilia: A Fly Geneticist's Swiss Army Knife". Genesis. 34 (1–2): 1–15. doi: 10.1002/gene.10150 . PMID   12324939.
  7. ↑ "GAL10". WikiGenes - Collaborative Publishing. Retrieved 8 April 2019.
  8. ↑ Traven, A; Jelicic, B; Sopta, M (May 2006). "Yeast Gal4: a transcriptional paradigm revisited". EMBO Reports. 7 (5): 496–9. doi:10.1038/sj.embor.7400679. PMC   1479557 . PMID   16670683.
  9. ↑ Fedor, Martha J.; Lue, Neal F.; Kornberg, Roger D. (5 November 1988). "Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast". Journal of Molecular Biology. 204 (1): 109–127. doi:10.1016/0022-2836(88)90603-1. PMID   3063825.
  10. ↑ Bitter, Grant A.; Egan, Kevin M. (30 September 1988). "Expression of interferon-gamma from hybrid yeast GPD promoters containing upsream regulatory sequences from the GAL1-GAL10 intergenic region". Gene. 69 (2): 193–207. doi:10.1016/0378-1119(88)90430-1. PMID   2853097.
  11. ↑ Bachhawat, Nandita; Ouyang, Qian; Henry, Susan A. (October 20, 1995). "Functional Characterization of an Inositol-sensitive Upstream Activation Sequence in Yeast: A cis-regulatory element responsible for inositol choline-mediated regulation of phospholipid biosynthesis". The Journal of Biological Chemistry. 270 (42): 25087–25095. doi: 10.1074/jbc.270.42.25087 . PMID   7559640.
  12. ↑ Ouyang, Qian; Ruiz-Noriega, Monica; Henry, Susan A. (May 1, 1999). "The REG1 Gene Product is Required for Repression of INO1 and Other Inositol-Sensitive Upstream Activating Sequence-Containing Genes of Yeast". Genetics. 152 (1): 89–100. PMC   1460607 . PMID   10224245.
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