BIOL 2960 Lecture 29: Lecture 29-31: Eukaryotic Transcription and Regulation

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Washington University in St. Louis
Biology And Biomedical Sciences
Biology And Biomedical Sciences BIOL 2960
Kunkel Barbara

Lecture 29-31 Saturday, April 22, 2017 11:52 PM EUKARYOTIC TRANSCRIPTION & REGULATION • Introduction to Eukaryotic Transcription ○ Clicker: These are true about both eukaryotic and prokaryotic transcription: ▪ Both use DNA sequences near the transcription start site to position the polymerase for initiation (promoters) ▪ Both have to "open" the DNA at the transcription start site ○ Gene expression changes in single-celled organisms allow the organism to adapt to different environmental conditions. Gene expression changes in multi-cellular eukaryotic organisms allow formation of different cell types as well as adaptation to different environmental conditions ○ Major differences: ▪ In prokaryotes, it's not compartmentalized, and transcription and translation happen at the same time essentially. In eukaryotes, transcription occurs in the nucleus ▪ In eukaryotes, there are RNA processing events before being transported to the cytoplasm, whereas in prokaryotes there is basically none ▪ ○ Features of Transcription in eukaryotes: ▪ Spatial separation of transcription and translation ▪ More complex transcriptional regulation: □ Three RNA polymerases □ General Transcription Factors (GTFs) □ Cis-acting elements (DNA regulatory sequences that bind proteins) are more varied and can be positioned in different configurations relative to the coding sequence ▪ Extensive processing of primary transcript destined to become mRNA in eukaryotes ○ Bacterial vs Eukaryotic Regulation ▪ Bacterial: Ground state is on. Template for transcription is protein-free. Activators enhance weak polymerase binding; repressors interfere with polymerase binding. Polymerase holoenzyme binds protein-free DNA. Promoters are DNA. ▪ Eukaryotes: Ground state is off. Template for transcription is chromatin which is inaccessible to RNA polymerase. Activators make chromatin accessible and/or promote the formation of a marked site on the DNA. Repressors interfere with transcription by blocking activators or by making chromatin less accessible. RNA polymerase binds to protein-DNA complexes which mark the DNA. Promoters (location where polymerase binds) are really protein-DNA complexes embedded in a chromatin environment. • Nucleosomes and Chromatin • Nucleosomes and Chromatin ○ The average chromosome is 5cm, whereas the nucleas is about 5-10 * 10^-4 cm, so this doesn't fit! ○ All eukaryotic DNA is packaged into nucleosomes. DNA is wrapped around a protein core made of 8 histone molecules: H3 H2 H2A H22 + (21 Linker) ▪ Each of the "flavors" of histone have a common histone fold structure: helix, turn, helix, turn, helix ▪ Two histone folds can combine together into a "handshake": H2A/H2B and H3/H4 shake hands ▪ H4/H3:H3\H4 handshake tetramer dimers form the core -- two handshakes bind to one another. Then, one H2B/H2A binds to the front, and one H2A/H2B binds in the back. ▪ The octamer core provides a cylindrical ramp along/around which the DNA can be wrapped. Properties of ramp: □ Main contacts are between histones and minor groove DNA determinants. A/T base pairs are favored at sites of interaction. This implies sequence-specific positioning of nucleosomes on DNA □ DNA is slightly underwound; 10.2 bp per turn compared to 10.5 bp of canonical B-DNA -- the ramp is not perfect! ○ Lysine: important player in gene regulation ○ Histone modification ▪ Histone amino-terminal "tails" are conserved but are not part of the "core" and are targets for covalent modifications that regulate chromatin structure and provide binding sites for proteins -- H3 is most important □ Modifications include:  Acetylation* ◊ Associated with activation  Methylation* ◊ Associated with both activation and repression, depending on which lysine is methylated  Phosphorylation  Addition of ubiquitin -- a small polypeptide □ These modifications are dispersed throughout the tail □ These modifications are dispersed throughout the tail ▪ Remember: DNA is acidic because of negatively charged phosphates ▪ Histones are basic because of abundant positively charged lysine and arginine amino acid residues. Lysines in amino terminal tails can be modified by acetylation, which neutralizes the positive charge. ▪ Methylation of H3K9 (the lysine (K) in the 9th position on the H3 tail) is associated with gene suppression ▪ Histone tail interacting protein classifications: □ Writers: covalent modification of histone amino acids  e.g. histone acetyltransferase (HAT) -- activates transcription □ Erasers: restore modified histone amino acids to unmodified form  e.g. histone deacetylase -- represses transcription □ Readers: bind modified histone amino acids  Chromodomain: can bind to methylated lysines  Bromodomains: can bind to acetylated lysines ○ Think of chromatin as a compact structure that needs to be made accessible for transcription, replication, and DNA repair • Gene Regulatory Structure ○ Focus on RNA Pol II, which is responsible for transcribing mRNA and snRNA ○ Protein binding sites: ▪ Promoter: region around transcription start site responsible for indicating where the enzyme should bind such that translation can begin at the +1 site □ Core promoter: set of sequences required to recruit General Transcription Factors and RNA Polymerase to non-chromatin DNA in a test tube -- does not function alone in chromatin environment, needs nearby sequences to complete promoter activity ▪ Enhancer elements: work from far away to increase transcription; they are needed for maximal promoter activity, and often involved in cell-type specific expression (for example, liver, muscle, or gut) □ Most human genes have multiple enhancers, spread over large distances, each responsible for activating expression of the gene at a specific time or place. □ Example: Sonic hedgehog gene, a developmental regulator, that has enhancers spread over more than 1 million base pairs -- 1 gene expressed at different places and times in the development of an embryo, so it is transcribed multiple times, but at certain points only particular enhancers are activated depending on the stage □ Human sonic hedgehog is related to the drosophila hedgehog gene, which codes for a signaling molecule used in development. It looks like a hedgehog, hence the name. This activates a signaling pathway for the activation of enhancers in other genes as well. • Quick overview of transcription: • Basal Transcription ○ Although a core promoter in chromatin is inactive, what do you need to transcribe it in the absence of chromatin? RNA polymerase by itself cannot bind to the transcription start site, need general transcription factors Core promoter elements are adjacent to the transcription start site, required to get RNA ○ Core promoter elements are adjacent to the transcription start site, required to get RNA polymerase onto promoter, sufficient in vitro on chromatin free DNA -- no need for other stuff, may be present in different combinations on individual promoters -- these do not bind to RNA polymerase directly; they allow GTFs to bind onto them; e.g. TATA box ▪ General transcription factors: TFIIA,B,D,E,F,H; required for basal transcription in
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