changes tertiary structure and enables it to fit into major groove of DNA site
CAP binding causes what to happen in the promoter DNA?
94 degree bend, stabilizes binding of RNA polymerase to the -10 and -35 regions of the lac promoter, increasing transcriptional rate
RNA transcription and DNA transcription factors are modular in domain and have separate bindings
What are the three domains of CAP:
1. ligand binding (cAMP)
2. DNA binding (promoter)
3. protein-protein interaction (binding between two monomers)
Palindromic DNA in operator is recognized by
lac repressor protein
Where does the lac repressor bind?
as a dimer to the operator
tryptophan leader sequence is found where in regards to operator?
In what phase of the bacteriophage lambda is cI repressor gene activated?
repressors bind to...
phage lambda: when repressor level is low in the cell, what operator site will it bind to first?
Binding to Or1 with the lambda repressor does what?
blocks transcription of the cro gene and recruits binding of a 2nd repressor to the weaker Or2 site, which recruits RNA polymerase to stimulate transcription of its own gene...the cI gene
as the level of phage lambda repressor increases, a 3rd repressor bind to Or3, which does what?
inhibits transcription of the cI repressor gene
When the level of lambda repressor drops, what becomes unoccupied?
OR3, and cI is activated to synthesize more repressor
lambda repressor is a monomer. true or false
recA can be induced by what?
UV light to degrade lambda repressor
protease that can degrade lambda repressor
enhancer assembles a complex, true or false
What does the ligand binding domain bind?
inducers, hormones, etc.
activating domain binds what?
other transcription factos and RNA polymerase II to activate transcription
DNA binding domain: binds to specific DNA motifs in the
promoters, enhancers, and LCRs
many different dna binding domains like:
where do helix-turn-helix (homeodomain) proteins bind?
major groove of DNA.
dimer-dimer proteins bind to ___________ sequences
What are some examples of helix-turn-helix proteins
CAP, lac, trp repressors of E coli
leucine has residues at every 7th residue. true or false?
cysteine and histidine can bind what kind of atom?
zinc, plays role in zinc finger motif.
zinc finger motifs are most common motifs in human gene.
nuclear proteins are transcription factors that work in the nucleus and bind to hormone responsive elements
hormone receptor is a zinc finger transcription factor
where do transcription factors bind?
to the enhancer or promoter
zinc dinger motifs bind to sites that are usually
steroid receptors of the nuclear receptor family are all....
do coregulators like coA, coR, co-activators, co-repressors bind DNA?
no, they are recruited by transcription factors and interact with basal promoter complex assembled at TATA box
coA has a DNA-binding domain: true or false
false, protein-protein binding domain
hormone receptors interact with what to assemble the transcription complex?
How does tamoxifen work?
binds to ligand site on estrogen receptor in place of estrogen, and the side group does not induce a conformational change, which blocks the ability of coactivators to bind and expression of genes will not be activate, since the estrogen receptor cannot activate the responsive genes
What does epigenetic modifications do?
modulate transcription of resident genes
how are stem cells differentiated?
passed through generations thanks to epigenetic modifications
what are operons regulated by?
activators and repressors
Is a repressor-binding site upstream of an operator cis-acting or trans-acting?
What does the lacI gene product due?
Called LacI, it is a repressor protein that binds to the operator when glucose is plentiful
Where is the operator in the lac operon?
between the promoter and transcription initiation site for LacZ gene
Allolactose binds to what in the lac operon?
it is an inducer. it binds the repressor which is bound to the operator
true or false: allolactos changes the conformation of the repressor.
true, and it dissociates from the operator DNA
lacZ encodes what, and what does it do?
beta-galactosidase, breaks down allolactose into galactose and glucose
Promoter-operator region of lac operon. What can bind here?
to the promoter, in order to increase binding strength of promoter to RNA polymerase.
True or false: when CAP binds promoter, transcription rate increases
what is CAP activity regulated by?
What is the activating ligand for CAP?
when there is high glucose levels in the medium, cAMP levels are...
low. since glucose=no lacz.
what interactions does CAP facilitate when binding RNA polymerase to promoter?
Protein-DNA and protein-protein interactions
When CAP binds to promoter DNA, a 94 degree bend occurs. This stabilizes binding of RNA Pol to what?
-10 and -35 regions of lac promoter
What are the three domains of CAP?
ligand binding (to cAMP), DNA binding (to promoter), and protein-protein-interaction (dimeric CAP)
T or F: lac repressor is a dimer
what are lac repressor's three functional domains?
protein-protein interaction (dimeric lacI), inducer-binding (allolactose) and DNA binding (operator)
amino acids can regulate transcription of their own operons. what is an example of this presented in class?
Tryptophan operon attenuation
The leader RNA in the 5'end of the polycistronic mRNA of trp can do what to attenuate levels of transcription?
form a terminator loop, a secondary structure of a stem loop followed by poly U.
the stem-loop structure stops what from proceeding?
the RNA polymerase
when tryptophan (Trp) levels are high, Trp forces formation of what, and activates what?
1. forces formation of terminator stem-loop in attenuato RNA
2. activates Trp repressor, which binds tightly to the operator
scarcity of trp-tRNA causes what?
ribosomes to stop at trp codons, which allows formation of the anti-terminator and RNA pol can continue
When ribosome passes quickly over trp-trp codons of leader mRNA, what forms?
lambda repressor binds to what site with the highest affinity?
Or1, which blocks transcription of the cro gene
When lambda repressor binds to Or1, what then happens?
cro gene transcription blocked, Or2 bound by second repressor, which recruits rna pol to transcribe cI gene
as the level of lambda repressor increases, what happens?
lambda repressor binds to OR3, which inhibits further transcription of CI repressor gene.
what does binding of Or1, Or2, and Or3 entail?
or1- cro gene blocked, recruits binding of lambda repressor to or2.
or2- recruits rna pol to transcribe cI repressor
or3- blocks transcription of cI repressor at high levels of lambda repressor
stem cells rely on what to differentiate?
histone (epigenetic modification)
Histones have a lot of basic amino acids. t or f
Histones do not bind dna in a sequence specific manner. t or f
How does transcription bind DNA when it's around histones.
recruits co-activator, HAT, which has histone acetyltransferase
What does HAT do?
acetylates lysine residues on histone tails, which recruit chromatin remodeling protein complex.
What is the repressor involved in histone regulation?
Histone deacetylases, HDACs
True or false: chromatin remodeling protein complex uses ATP.
methyltransferase on DNA methylates what base most often?
Cysteine, in CpGs
CpG islands are found most often in what?
CpG islands in promoter and upstream regulatory sequences
Methylation is restriction to CpG, true or false
false, also CpApG
when genes are imprinted, what happens?
they are inactivated
when genes favoring fetal growth are expressed only when are expressed from the
When DNA at the barrier/insulator element is methylated, what happens? (or doesn't happen)
it can't bind CTCF, rendering the insulator non-functional.
IGF2 stands for what?
Insulin growth factor 2, and plays a part in Beckwidth-Wiedmann syndrome (BWS)
BWS involves what mutations?
beckwidth-wiedmann syndrome, mutations in the barrier/insulator DNA
normal barrier/insulator DNA can bind what?
In normal barrier/insulators that have bound CTCF, what doesn't happen?
The IGF2 can't interact with the enhancer on the 3' end, so it becomes hypermethylated and inactive
In normal, parental chromosome 11, what happens with the barrier DNA?
it is methylated and cannot bind CTCF, so it's nonfunctional. the IGF-2 interacts with the 3'enhancer and it is hypomethylated/activated
IN BWS patients, what is wrong with the maternal chromosome, if anything?
The barrier DNA is mutated/ deleted, so it can't bind CTCF and thus the IGF2 gene interacts with the 3' enhancer and gives a double dosage of IGF2 since that father has methylated barrier DNA.
What can double dosage of IGF-2 manifest as in patients?
organ overgrowths, mental retardation, cancer susceptibility
Mutations in cis element, B globin promoter, will cause what?
beta-thalassemia, since it does not EKLF (erythroid specific transcriptional activator)
Mutations in transcription factors like _______________ can cause cancer, specifically breast cancer, and kidney cancer
p53, BRCA1, WT1
leukemias can be caused by what problems with transcription factors?
dysregulation due to chromosome translocation
epigenetic therapy is used to
use DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors as potential cancer drugs. Both drugs target tumor suppressor genes that have been abberantly silenced by epigenetic modifications
What happens when there is mutations in the WT1 gene?
Wilms' tumor, WT
Wilms' tumor is what kind of tumor
kidney tumor from mutation in WT1, zinc finger transcription factor
Mutations in zinc fingers and some splice isoforms are bad because
they affect DNA binding strengths of the zinc fingers, cause tumors
Many leukemias are caused by
C-myc translocated to next to IgH gene, what happens?
igH has powerful promoter
HIV hijacks what?
transcription factors and co-factors deprives transcription for host genes
98% of non-coding RNAs are transcribed by RNA polymerases II and III. what do they do?
1. small non coding RNAS (22-25): miRNA and siRNA that act post-transcriptionally to regulate mRNA stability and translation.
2. lncRNAS (>200) long non coding RNAs, new, regulate cellular proccesses in development, differentaiation
siRNA and miRNA are transcribed from
intergenic regions, or invading viruses
dsRNA gets chopped up by
dicer and drosha
dsrna is loaded into a RISC, which is
RNA induced silencing complex
siRNA and miRNA are delivered well why?
small in size
why are siRNA/miRNA have potential for gene therapy?
they are small, easy to get into cell, and target specific genes
acetylation, methylation, pshophorylation, all happen in what?
What is the inducer in lacZ operon?
Activators can bind to enhancers or promoters. What is lacZ's promoter's activator?
Catabolite gene Activator Protein, or CAP. it is regulated by cAMP
What happens when cAMP binds CAP?
It changes its comformation of (CAP) and enables it to fit snugly in major grooves of DNA motif.
high cAMP, means
low glucose, since glucose is an inhibitor of adenylcyclase
CAP is a dimer, true or false
Where does the repressor sit on the lac operon?
on the operator
Is the lac repressor also a dimer?
What is the stem-loop in the terminator RNA loop consist of in trp operon?
lots of GCs followed by poly US
What binds to the repressor in order for it to bind to the operator of trp operon?
What is the activator for the trp repressor?
What do high Trp (tryptophan) levels in a cell accomplish in regards to the trp operon?
-forces formation of terminator loop
-binds to trp repressor, activating it to block RNA pol by binding to operator
what happens when Trp is high in regards to transcriptional speed?
high Trp= lots of trp-tRNA, so ribosomes translate through Trp codons in leader sequence quickly, which favors formation of a terminato loop that pulls RNA pol off DNA
cI repressor gene activated in viral DNA when
dormant phase to suppress infective viral genes
operators Or1, Or2, Or3 contain what kind of DNA sequences?
each gene in eukaryotic cells has its own promoter and enhancer sequences: t or f
true, each gene has its own promoter and enhancer sequences
Eukaryotes need an active transcription complex to start transcription. what are its general components?
- cis elements: promoter, enhancer
- enhancer activators, transcriptional activators on promoter, co-activators, mediator recruited by promoter
Eukaryotic transcription factors have modular domains. what are they? (3)
1. ligand binding domain (inducers, hormones)
2. Activating domain: binds to other transcription factors and RNA pol II to activate transcription.
3. DNA binding domain- binds to specific DNA motifs in promoters, enhancers, LCRs
what are the 4 DNA binding domain examples?
3. leucine zipper
4. zinc finger
helix-turn-helix motifs bind to dna where
in DNA palindromic sequences in the major groove. This includes CAP, lac, and trp repressors
dimer (required for function) that regulates immune system genes. It contains hydrophobic residues positioned towards one side of the helix
bZIP, basic leucine zipper
regulates cell division genes. rich in leucine residues that provide dimerization motif. the zipper is made of leucine and it is the stem of the "Y". The hydrophobicity of leucine at every 7th reside causes indigitation with the strand
is zinc finger a dimer?
yes, held in "finger" arrangements by Zinc coordination
What do steroid hormone receptors bind to DNA through?
zinc finger domains
how does a steroid hormone affect transcription?
1. diffuses into cytoplasm
2. binds cognate receptor (zinc finger)
3. zinc finger binds to DNA and stimulates transcription
What is an HRE, or hormone response element?
a short region of DNA in the promoter that binds transcription factor zinc finger and steroid hormone.
steroids can form dimers, homodimer or heterodimers. this can cause what in regards to transcription?
transcription can start and another turned off in response to the same heterodimer
Hormone receptors interact with what to assemble the transcription complex for DNA?
with co-activators or co-repressors, which do not directly bind DNA.
Co-activators/repressors form a bridge between what in hormone-response synthesis?
They attach to the hormone receptor, which in turn attaches to hormone-response elements (HRE). CoA/ CoR forms a bridge between transcription factors located at the enhancer (potentially far away) and the promoter (with TFIID bound to TATA box of RNA Pol II)
Breast cancer cells need estrogen for transcription and growth. how does tamoxifen block this?
-Tamoxifen antagonist of estrogen
-competes for ligand spot on estrogen receptor
-once bound to estrogen receptor, co-activator cannot bind to estrogen receptor
- no bridge linking receptor and TFIID promoter complex at TATA box
Which histones form the octamer core of nucleosomes?
H2A-H2B and H3-H4, two dimers each
what are some epigenetic modifications of histone core?
acetylation, methylation, phosphorylation, that modify transcription of genes
What are the sites of epigenetic modifications in histone cores?
Amino terminal tails sticking out
What are the steps of chromatin remodeling via transcription factor?
1. transcription factor binds to promoter/enhancer in a gene packaged in nucelosomes
2. coactivaor that TF recruits has histone acetyltransferase activity (HAT)
3. Co-activator acetylates lysine residues in tail, which recruit chromatin remodeling protein complex with ATPase activity.
4. sites exposed through ATP-dependent remodeling and transcription factors like RNA pol II bind and activate.
5. HDAC (histone deacetylases) associated with co-repressors deacetylate the histones, close down, trun off transcription
How are coactivators and Co-repressors involved in chromatin remodeling?
They have coactivator-HAT and corepressor-HDAC activity, which
HAT: acetylates lysine resides in N terminus which recruit CRPC
HDAC: turns off everything
where are most CpG islands found?
in promoters and upstream regulatory sequences.
What does methylated DNA do?
recruits HDACs and other co-repressor proteins to close down chromatin structure and repress transcription
When a promoter for erythroid cells is hypomethylated, what happens?
globin genes turn on
acetylated histones: active genes, loose histones. t or f
how are imprinted genes inactivated?
methylation of CpG islands in a parent-specific manner
what are some examples of diseases caused by imprinting error
Beta thalessemia is caused by what
errors in the B-globin promoter, cannot by EKLF
mutations of what protein (trans protein) are found in 50% of cancer cases
Mutations in the WT1, a zinc finger transcription factor, cause
childhood kidney cancer
DNA methylation inhibitors and histone deacetylase inhibitors are potential cancer drugs why?
They target tumor suppressor genes that were silenced by DNA methylation and HDAC
Wilms' tumor is caused by
defect in WT1 gene encoding zinc finger transcription factor
acute promyelocytic leukemia is caused by chromosomal transloaction of steroid hormone receptor gene and dysregulation of zinc finger transcription factor-
siRNAs and miRNAs are what kind of noncoding RNA
what does siRNA and miRNA act on
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