Eukaryotic Transcription Regulation

Vocabulary:

• Cell-type specific expression: Different cells may express very different sets of genes, despite containing the same DNA, allowed for by gene regulation

• Chromatin: While DNA is in this structure, transcription, replication, and DNA repair are all inhibited

• Nucleosome: The fundamental subunit on chromatin, 2 turns of DNA wrapped around 8 histones, making an octamer

• Histone: A protein that DNA wraps around, small and positively charged, largely Arg and Lys, bind to DNA and are highly conserved

• Histone H1: The protein that binds to linker DNA between nucleosomes forming the macromolecular structure known as the chromatin fiber

• Chromosome territory: Within a nucleus in interphase, chromosomes will occupy discrete domains and stay separate from other chromosomes

• Interchromosomal domain: Channel between chromosomes that contain little or no DNA

• Transcription factories: A feature within the nucleus that may contribute to gene expression, contain most active RNAP and transcription regulatory molecules, they are dynamic structures that form rapidly and disassemble upon stimulation and repression of transcription. They are at the edge of chromosome territories and move into the adjacent interchromatin compartments. They house RNA processing machinery and are contiguous with nuclear pores.

• DNase I hypersensitivity: Results in more open chromatin configuration states, upstream of the transcription start site

• Histone modification: The addition of acetyl groups to histone proteins and histone protein tails, and the addition of methyl groups to histone protein tails and DNA

• “Closed” chromatin: An inhibitory conformation of DNA, where DNA is tightly associated with histones

• DNase I: Digests DNA to varying degrees depending on the conformation

• H2A.Z: Affects nucleosome mobility and positioning on DNA, found more at promoters and enhancers

• Histone modification: Involves covalent bonding of functional groups onto N-terminal tails of histones

• Histone acetyl transferase enzymes (HATs): Associated with increased transcription, catalyze the addition of an acetyl group to a histone to make the charge more positive and cause the histone to bind less strongly do DNA

• Histone deacetylase (HDAC): Remove acetyl groups from histone tails, associated with a decrease in transcription

• Chromosome remodeling: Involves the repositioning or removal of nucleosomes on DNA, multi-subunit enzymes use the energy of ATP hydrolysis to remove and arrange nucleosomes. Directed by binding to transcription factors, modified histones, or methylated DNA

• DNA methylation: Enzyme mediated addition or removal of a methyl group to DNA, done by a methyltransferase, occurs most commonly at position 5 of cytosine, causing it to protrude into the major groove of the DNA helix, can decrease gene expression, tissue specific

• CpG Island: A region with many methyl groups on DNA, occurring near the promoter

• Silenced DNA: Happens when there are many methyl groups attached to DNA, this is what happens to the Barr body

• Cis-acting sequence: Located on the same chromosome as the gene it regulates, required for accurately regulated transcription on genes, either a promoter, enhancer, or silencer

• Promoter: A nucleotide sequence that serves as a recognition site for transcription machinery, adjacent to regulatory genes and critical for transcription initiation

• Core protomer: A protomer that determines accurate initiation of transcription, minimum required for accurate initiation of RNAP II, around 80 nt long, has the transcription start site (TSS)

• Proximal promoter elements: Modulate efficiency of transcription, around 250 nts upstream of the TSS, have binding sites for sequence specific factors that modulate the efficiency of transcription

• Focused promoters: Specific transcription initiation at start sites, major type of initiation for lower eukaryotes

• Dispersed promoters: Direct initiation from several weak transcriptional start sites located over 50-100 nt region

• Enhancers: Located on either side of a gene, or within, important for reaching max levels of transcription, cis elements, orientation/position independent, confer time/tissue specific gene expression, modular and have many binding sequences that additively enhance effects

• Green Fluorescent Protein (GFP): Emits a green fluorescence when excited by blue light, used in reporter genes, can show effect of an enhancer on transcription

• Insulators: Found between the enhancer and promoter for a nontarget gene, limits the extent of enhancers, enable and block enhancer/promoter interactions

• Silencer: Can be on either side of a gene, or within, acts as a negative region of transcription, represses levels of initiation, act as a tissue/time specific way to control gene expression, binding site for repressor proteins

• Transcription factors (Tx): Regulatory proteins involved in transcription, target cis-acting sites

• Activator: A kind of Tx that increased transcription initiation

• Repressor: A kind of Tx that decreases transcription initiation

• Human metallothionein IIA gene (hMTIIA): A protein that binds to heavy metals and protects cells from toxic effects of high levels of them, protects cells from oxidative stress, can be upregulated

• DNA-binding domain: Part of a Tx, binds to specific DNA sequences in the cis site, forms hydrogen bonds with DNA, fits into the major groove

• Trans-repressing domain: Part of a Tx, activates/repressed transcription by binding to other transcription factors or RNAP

• Helix-turn-helix (HTH): Part of a DNA-binding protein, present in both eukaryotic and prokaryotic Tx, monomer has 2 alpha helices, binds to the major groove

• Zinc finger: Part of DNA-binding proteins, uses Zn to stabilize structure, has many protrusions that make contact with the target molecule

• Basic leucine zipper (bZIP): Part of DNA-binding proteins, allows for protein-protein dimerization, has a region that mediates sequence specific DNA properties, holds together 2 DNA binding regions

• Pre-initiation complex: Formed by the assembly of proteins in a specific order, provides a platform for RNAP II to recognize the TSS. Formed by TFIID binds to the TATA box via TBP to start forming this, followed by the addition of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and the final mediator

• Coactivator: Interacts with proteins and enables an activator, form an enhanceosome

• Enhanceosome: Formed by coactivators, interacts with the transcription complex

• GAL gene system: 4 structural and 3 regulatory genes, products transport galactose into the cell to metabolize it, inducible, genes not transcribed without galactose and positively regulated by galactose

• GAL4: The regulatory gene that activates proteins that regulate GAL structural product transcription

• GAL4 Protein (GAL4p): Encoded by GAL4, negatively regulated by Gal80p, has a DNA binding domain that recognizes and binds to sequences in UAS

• ENCODE: The encyclopedia of DNA elements project, launched in 2003 to identify all functional DNA sequences and determine how elements regulate expression in humans, determined less than 2% of the genome is proteins, with over 80% being regulatory of these

• Enhancer elements: Contain promoter elements that initiate transcription

• Enhancer RNA: A genomic sequence that encodes enhancer RNA, often contain promoter elements that initiate transcription from the enhancer and extend outward for several kilobases

• Genomic wide association study (GWAS): Identifies genetic variations that are significantly enriched in the genomes or patients with a particular disease and are not found in those without the disease, can evaluate single base pair changes or SNPs

With eukaryotes, regulation can occur during chromatin remodeling, splicing, nuclear export, mRNA stability, mRNA localization, translocation, and protein modification. Genes are on chromosomes, and are combined with histones

Within a chromosome, structure is continuously rearranging, transcriptionally active genes are cycled to the edges of a chromosome’s territory

SWI/SNF binding will cause the nucleosome to arrange in a way where DNA histone contacts may be loosened (allowing the nucleosome to slide along the DNA and expose regulatory regions), the path of DNA around a nucleosome core particle may be altered, or components of the core nucleosome particle may be rearranged such as swapping in and out variable histone proteins

A protein will bind to an insulator and induce the formation of a DNA loop, which will allow specific enhancer-promoter interactions and not others

Galactose metabolism is inducible, transcription is activated by GAL4 in response to galactose, where GAL4p binds to the UAS site and controls transcription levels, UAS has the properties of an enhancer, as it is a regulatory sequence lying upstream of the gene.