Hipfner 1 Flashcards
What is the sigma factor?
In prokaryotes → subunit of RNA polymerase → binds to -35/-10 promotor sequences to properly position the holoenzymes at the trascription start site
*Default transcription state of prokaryotes → ON
What is an inducer?
When the presence of an effector leads to increased gene expression its called an inducer → through allosteric transition, it binds to TF
Inducer binds to repressor → prevents it from binding to DNA
Inducer binds to activator → makes it bind to DNA
What are the components of the Lac operon?
I → codes for repressor protein → binds to the operator → blocks transcription
P → promotor
O → operator
structural genes:
Z → Beta-galactose
Y → Permease
A → lacA
What is the effect of lactose on the Lac operon regulation?
Lactose binds to repressor protein (encoded by I gene) → when bound to lactose, operator can’t bind DNA → can’t block RNA pol from transcribing → GOOO
What are uninducible and constitutive mutants of the lac operon?
Uninducible mutant → can’t make LacZ AND LacY in presence of inducer IPTG (form of lactose)
Constitutive mutant → make both LacZ AND LacY even in absence of IPTG
How does glucose act to regulate the lac operon?
High glucose inhibits/prevents ATP → cAMP
Low glucose allows ATP → cAMP
CAP-cAMP binds to the promotor → facilitates RNApol binding to P → increases transcription (would still be on without, but a lot less)
What gene codes for CAP?
CAP = Catabolite Activator Protein → CAP-cAMP binds to promotor of lac operon with cAMP
crp gene codes for CAP
What are features of prokaryotic cells?
ex: E.coli
- no nucleus → transcription and translation can occur simultaneously
- Single circular chromosome → DNA not wound up in nucleosomes as in chromosomes
- intronless-genes → no splicing
- genes encoding for enzymes involved in same metabolic process often organized into operons
What are features of eukaryotic cells?
- DNA is wound up in nucleosomes → much harder for RNA pol and TF to bind to their target sequences → default = OFF state
- Genes have introns → splicing required
- Presenc of a nucleus → transcription and translation are separated + extra step to export mRNA to the cytoplasm
What are the cis-acting DNA sequences for transcription initiation?
- core promotor
- promotor-proximal elements
- enhancers/silencers
What are the trans-acting proteins involved in transcritpion initiation?
- General TF
- Common TF
- Cell/tissue-specific TF
- transcription cofactors → do NOT have DNA-binding domains (TFs do)
How do specific TF regulate transcription?
- Expressed in specific cells-types/tissues/times
- Binds to enhancer sequences → distance independent manner
- Bind DNA through DNA-binding domains
Influence transcription by:
- Interacting with transcription apparatus → direclty or indirectly (enhanceosome)
- Influence chromatin structure → directly or indirectly
What different domains do eukaryotic TFs have?
N - DNAbindingDomain - DimerizationDomain - Ligand-bindingDomain - Activation/RepressionDomain - C
Ligand-binding domain → allosteric switches
Activation/represion domain → interacts with other components of the transcription machinery
*Cofactors can have similar domains, but NO DNA-binding domain
*Same gene can be regulated differently in different tissues because of different enhancer regions → different TF are expressed in different cell types
What are the different components of the yeast GAL system?
What is the use/importance of the yeast GAL system?
Glucose and Galactose are almost identical in structure → only difference is the orientation of -OH group on 4th carbon
Most living cells → glucose is preferred sugar in the metabolism → GAL converts galactose to glucose-1-P for energy
*GAL only works in presence of galactose
What is GAL4?
GAL4 is a transcriptional activator → binds to enhances called “Upstream Activator Sequences” (UAS)
Has Activation domain + Dimerization domain + DNA-binding domain
GAL4 activation domain can bind to protein of the trascriptional machinery:
- Help recruit RNA pol II
- Helps recruit chromatin modifying proteins
- Can function in many eukaryotic cells (insects, humans, etc.)
What is comon of GAL1, GAL2, GAL7, GAL10?
They are all galactose-responsive enzyme regulated by GAL4, GAL80 and GAL3
Few UAS upstream from each GAL enzyme genes → each have their own promotor, distance independent
What are the roles of GAL80 and GAL3 in the GAL4 system?
(Which 2 words qualify GAL3?)
GAL80 binds to the GAL4 activation domain to prevent it from interacting with other proteins
binding of galactose and ATP → changes structure of GAL3 → binds to GAL80 → removes GAL80 from GAL4
*GAL3 = sensor and inducer
What are the roles of insulators?
Enhancer-blocking insulators restrict action of enhancers to specific genes
Insulators → DNA sequences specifically bound by insulator proteins (CTCF is an example of insulator proteins)
→ helps to organize chromosomal DNA into specific sub-domains TAD (Topologically associating domains)
CTCF works with cohesin which makes loops → TAD
Enhancers can only act on promotors inside a TAD
What is the role of SWI/SNF?
Transcriptional coactivators → don’t bind DNA, but get recruited by enhancer-bound TF → remodel chromatin/nucleosomes locally
use ATP to reposition or remove single nucleosomes → expose binding sites
*Not epigenetic transformations/histone modifications
What are the 4 possible histone tail modifications?
What are the general effects of these modifications?
Covalent modifications:
- Acetylation
- Methylation
- Phosphorylation
- Ubiquitination
→ Can alter charge → affect affinity for DNA
→ Can affect binding of other regulatory proteins → histone code
What is the effect of histone acetylation?
It relaxes chromatin by neutralizing positive charge on lysine → associated euchromatin → increased transcription
Also creates binding sites for histone cose readers → help promote activation
HAT enzymes → writers → catalyze acetylation
*transcriptional (co)activators often have this activity
HDAC → erasers → remove acetylation
*transcriptional (co)repressors often have this activity
How/Where do histone methylations occur?
- On lysine or arginine residues
- Catalyzed by histone methyltransferase (HMTase) enzymes
- Doesnt’ change the charge of histone → addition of 1, 2 or 3 methyl groups on N+
*Histone marks generally associated with gene silencing and act as signal to recruit specific readers
What is the effect of H3K9Me?
Methylation (speciifcally) H3K9Me promotes heterochromatin formation and tends to SPREAD in chromatin
1. HP-1 (Heterochromatin Protein 1) binds methylated histones, H3K9Me
2. HP-1 promotes heterochromatin formation by recruiting additional HMTase (HMTase binds to HP-1 which binds to H3K9Me)
3. HMTase methylates neighbouring nucleosomes, etc.