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.
What is responsible for stopping the spread in heterochromatin?
Barrier insulators (DNA sequences)
HATs bind to barrier insulators → counteracts spreading of heterochromatin (of methylation)
What is the definition of Epigenetic? Is it inherited?
It is the study of heritable traits that cannot be explained by changed in DNA sequence
Many modification to DNA and chromatin structure are inherited → passed to daughter cells (somatic) and future generations
During DNA replication, existing nucleosomes are disassembled → 1/2 to each daughter strand
Each nucleosome of the daughter strand reassembles as 1/2 old + 1/2 newly synthesized
Old histones with midifications (methylation) direct modification on new histones by recruiting readers and writers
What is the white gene in Drosophila eyes?
white gene codes an ATP-binding cassette (ABC) transporter that carrier the precursors of the red and brown eye color pigment into the developing eye
*Called that way because a mutation in it causes a white eye phenotype
What is position-effect variegation?
Give an example.
Epigenetic silencing by heterochromatin spreading:
Results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition
Ex:
In WT drosophila, between WT gene and Centromeric heterochromatin, there is a barrier insulator sequence. When inversion mutation → barrier insulator is the other side to the white gene → heterochromatin spreads on the white gene in some of the cells → come white, some red pigment cells
Which proteins can affect spreading of heterochromatin when mutated?
- Loss-of-function of HAT → spreading of heterochromatin → more white pigment cells
- Loss-of-function of HMT → less spreading → more red pigment cells
- Loss-of-function of HP-1 → less spreading → more red pigment cells
How might higher-order structures of chromatin activate, rather than repress transcription?
Through the formation of topologically associated domains (TADs), enhancer regions separated from promotors can easily find it in 3D dimensional space
What are 2 general mechanisms by which histone acetylation affects transcription?
- Histone acetylation reduced chromatin compaction by neutralyzing positive charges on lysine residues
- Creates binding sites for histone code readers (recognized by general TF and chromatin remodeling comlpexes (by bromodomains))
By what are 2 mechanisms could histone acetylation levels increase at a gene promotor?
By increased activity of HAT
By deacreased activity of HDAC
How woud you modify a transgenre so that its expression was not affected yby position-effect variegation?
*Transgene’s expression varies depending on state of chromatin at localization of insertion
By flanking the transgene with barrier insulator element sequences, prevent local chromatin structure at the site of insertion from affecting transcription of the transgene
For position-effect variegation to have been discovered, why is it critical that the white gene is on the X chromosome?
Because male flies only have 1 X chromosome, the chromosomal inversion that caused variegation of the white gene only had to happen once to produce an eye phenotype
*Would have had to happen twice in the same cell in female to have a phenotype
How might a co-repressor block transcription by RNA pol II?
Might bind a TF bound to an enhancer → block its ability to interact with TFIID, mediator or RNA pol II by blocking the activation domain
