VL 19 (George Soultoukis) Flashcards
Gene expression regulation levels
-
Activation and initiation level
–> Changes in local chromatin structure
Transcription initiation and elongation -
Nuclear mRNA processing
–> Capping, adenylation
–> Termination
–> (alternative) splicing -
mRNA export from nucleus
–> Transport to cytoplasm -
Translation of mRNA
–> Cytoplasmic, ribosome-mediated
5- Degradation
–> Performed by RNases
How is a gene turned on?
Activation and Initiation level
- A gene is turned on by initiation
- How does a TF bind to its target in a heterochromatin region?
–> Histones are temporarily removed during replication
–> Recruitment of histone modifiers / chromatin remodelers
–> Competition between TFs and nucleosomes
- Chromatin remodelling complexes alter chromatin state
–> Two simple, stable ground states:
Promoter occupied by histones
Promoter occupied by basal transcription machinery
–> Changes = chromatin remodelling
–> Histone reposition requieres energy
–> Complexes contain ATPases
Chromatin remodelling complexes
Chromatin Remodelling complex
* Complexes of 2 -> 10 subunits
Method:
1. Sliding of nucleosome
2. Nucleosome “spacing”
3. Nucleosome “displacement
- Remodeling consumes ATP
- Remodeling complexes contain ATPase subunits
- ATPase subunits determine remodeling complex functionality and classification
- Classification of remodelling complexes relies on type of ATPase and its subunits
Nucleosome Organization or Content Can Be Changed at the Promoter
- In general, remodeling complexes do not bind specific DNA targets but need to be recruited by activators or repressors:
- Sequence-specific activators or repressors bind upstream a target gene sequence
- Remodelling complex is recruited locally and binds to repressor or activator
- Remodelling complex remodels chromatin and displaces histone octamer
- Inorganic phosphate (Pi) is an essential nutrient for growth
- Saccharomyces cerevisiae a.k.a. yeast
- During phosphate starvation, yeast cells activate the phosphate signal transduction (PHO) pathway controlled by the Pho5 promoter
- Pho4 responds to phosphate starvation by displacement of nucleosomes
- Nucleosome displacement requires action of ATPase SWI/SNF + INO80
- Typically: 200 bp upstream of TSS in RNA PolII promoters that are nucleosome free
- Flanked by H2AZ-containing nucleosomes
Explain histone acetylation
- Newly synthesized histones are acetylated at specific sites
–> Deacetylation occurs after incorporation into nucleosomes - Acetylation is associated with activation of gene expression
Acetylation can occur locally or globally (e.g. on sex chromosomes)
–> HATs: Histone acetyltransferases
–> HDACs: Histone deacetylases
–> Group A HATs: act on chromatin
–> Group B HATs: act on newly synthesized histones (cytosolic)
Explain Histone and DNA methylation
DNA methylation:
* methylation of DNA and specific sites on histones inactivates chromatin.
* associated with transcriptional inactivity (CpG islands)
Histone methylation:
* Acetylation (COCH3) of histones activates chromatin;
* Activity/inactivity depends on specific site of methylation
–> Lysine (K) and Arginine (R) residues may be methylated
–> Lysine can be mono- to tri-methylated in H3 (multiple), H4 (one site)
—> Arginine mono- or di-methylated in H3 (3 sites), H4 (one site)
–> Large number of possible combinations
- Histone methyltransferases: HMTs/KMTs
–> KMTs contain SET domain - Lysine demethylases: KDMs
- Methylation of histones and DNA are connected in a reinforcing cycle
–> H3K9 methylation recruits protein HP1 which in turn targets DNA methyltransferase (DNMT)
–> Methylated CpG islands in turn recruit HMT and HDAC complexes
Histone Phosphorylation
- All histones can be phosphorylated
- Histone phosphorylation (PO4) associated with:
–> transcription
–> repair
–> chromosome condensation
–> cell-cycle progression
What are transcriptional activators?
- Activators stimulate transcription frequency
- Types of activators:
–> True activators
–> Direct or indirect contact (via a co-activator) with basal transcription apparatus
–> Anti-repressors
–> Act on chromatin, recruit chromatin remodelers
–> Architectural proteins
–> Bend DNA in order to facilitate or prevent protein complex formation
Modular architecture of transcriptional activators
- Activators interact with the basal transcription machinery
- Often: independent DNA binding and transactivation domain
–> DNA-binding domain dictates site of action
–> Activation domain determines mode of action - Prerequisite for Two-Hybrid Assays
Activators interact with the basal transcription machinery
- Typically interaction with:
–> TFIID, TFIIB or TFIIA
–> Activators stimulate the assembly of the basal transcription machinery - TAFs in TFIID interact with different activators, resulting in improved binding of TFIID or other basal components to TATA Box, or increased phosphorylation of CTD
- Acidic activators:
–> Negatively charged, stimulate TFIIB binding to basal apparatus - Stepwise assembly of several basal TFs, activators and coactivators
- Joined by large RNA polymerase holoenzyme preassembled with further activators
What is the mediator complex
- Yeast: RNA polymerase + large 20 (21) subunit complex (Mediator)
- Necessary for transcription of most yeast genes
–> Homologues in multicellular eukaryotes - Mediates effects of activators, transmits activating or repressing effects to CTD
DNA binding domains
- Different types of DNA-binding domains
- DBD-type often used for classification of TFs
- Zinc finger motif
–> Cys2/His2 finger: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His
–> Cys2/Cys2 fingers found in steroid receptors
Helix-turn-Helix
- Two α-helices that form a site that binds to DNA, one fitting into the major groove of DNA and the other lying across it.
- C-terminal alpha helix contacts major groove, middle helix lies across, N-terminal arm in minor groove
- Closely related: Homeodomain TFs
Leucine zipper motifs
- Amphipathic alpha helix
- Leucine in every 7th position
- Allows protein dimerization via interactions between hydrophobic surfaces of two Leucine zipper proteins
- Adjacent positively charged basic region makes DNA contact (bZIP)
Nuclear mRNA processing
5’ mRNA capping
- The 5’-ends of all eukaryotic pre-mRNAs studied so far are converted to cap structures
- The cap provides structural stability and various functionalities to the mRNA molecule
- Cap is required for mRNA export to the cytoplasm
- Caps influence translation initiation and splicing of the first intron
- The cap is bound by ‘cap-binding’ proteins, and provides resistance to mRNA exonuclease degradation
- The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II.
