Human Gene Transcription Flashcards
What is an example of a nonspecific protein?
pre-initiation complex
What are examples of a nonspecific element?
- TATA sequence
- nonspecific DNA sequences
How is transcription activated at the right time at the right place?
Specific proteins and elements
What subunit of TF2D binds to the TATA box?
TBP
Why is the H3K4 methylation needed?
- the TATA box is only 4 NT
- SSDBP (Sequence specific DNA Binding Proteins) recognize ~10 NT
What are the characteristics of nonspecific proteins?
- termed GTFs
- have RNA polymerase
- Facilitate positioning RNA Polymerase II to the proper start site for transcription .
- Assemble at the TATA box.
- Function in all RNA Polymerase II promoters.
- Are represented by TFIIX, where X can be any of A-H, each a different protein
Where does TF2H phosphorylate RNA polymerase 2?
on serine 5 of the carboxy terminal domain -> starts transcription
What is the carboxy temrinal domain a repeat of?
Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7
What starts the PIC?
TBP & TAF (TF2D)
What recognizes the TATA box?
TF2D
What unwinds DNA at transcription start point & phosphorylates Ser5?
TF2H
TF2D is composed of what 2 subunits?
TBP & TAFs
What subunit of TF2D binds directly to DNA & which does not?
- TBP -> binds
- TAF -> DOES NOT
What is the role of TAFs?
bind seuqence specific transactivators
What is developmental eukaryotic transcription?
transcription of different genes in different tissues or at different developmental stages
What is inducible eukaryotic transcription?
transcription of genes in response to a hormone
What are the 2 examples of developmental eukaryotic transcription?
- global switching = biochemical change from fetal to adult hemoglobin
- heart morphology = morphological change
How do transavtivators recruit TF2D to the TATA box?
by binding to the TAFs
What is the function of the transactivation domain?
- contacts other proteins, mainly TAFs
- helps TF2D stabilize the PIC
What is the function of the binding domain?
binds a specific DNA sequence
What is a consensus sequence?
general sequence that works for many different genes
The more transactivators in DNA looping to stabilize PIC = …
more likely for successful transcription
Do co-activators or repressors have a DNA binding domain?
no
How do co-acticators interact with DNA binding proteins?
- Most recruit HATs or contact the PIC
- Same role as transactivators; however, coactivators don’t bind a DNA sequence but rather bind to a DNA protein that binds to the DNA sequence
- Interact with DNA binding proteins
What are the characteristics of co-repressors?
- interact with DNA binding proteins
- recruit HDACs
- DO NOT bind DNA
What is an example of a classical repressor?
Rb protein
What 2 things interact with DNA but do not bind to DNA itself?
co-activators & co-repressors
What is the humanonco protein?
MYC
What are the 7 types of biochemical mechanisms that activate transactivators?
- protein synthesis
- ligand binding
- covalent modification
- addition of 2nd subunit
- unmasking
- stimulation of nuclear energy
- release from membrane
From the pituitary hormone deficiency article, what is it caused by?
heterozygosity for two novel mutations in the POU domain of the POU1F1 gene
From the pituitary hormone deficiency article, what is required for the transcription of the GH-, PRL-, and TSH-genes in the anterior pituitary?
transcriptional activator POU1F1
From the pituitary hormone deficiency article, several mutations in the gene encoding POU1F1 resulted in what?
combined pituitary hormone deficiency involving GH-, PRL-, and TSH
From the pituitary hormone deficiency article, patients with that phenotype can have what type of mutations?
either a dominant negative mutation in codon 271 (R271W) or are homozygous for a recessive mutation in the POU1F1 gene
From the pituitary hormone deficiency article, what did the boy described have?
- 1-bp deletion frameshift mutation (747delA) which leads to a nonfunctional truncated protein lacking the entire DNA binding domain of POU1F1
- a missense mutation in the C-terminal end of POU1F1(W193R)
From the pituitary hormone deficiency article, is the POU domain a type of DNA binding domain or type of transactivation domain?
POU1F1 is a transcription activator for GH, PRL, and TSHs in the anterior pituitary, the mutation describes causing a complete lack of DNA binding domain in POU1F1 meaning that the POU domain is a type of DNA binding domain
From the pituitary hormone deficiency article, “dominant negative mutation”, mentioned above in the case, means that the protein with this mutation would prevent transcriptional activation even if the protein encoded by the other allele was a wild-type protein. Describe a molecular mechanism that would account for this effect.
- Being a dominant negative mutation allows for the alleles to be heterozygous for the trait, but for the trait to still be expressed.
- Wild-type means a normal allele aka a functioning POU domain
From the pituitary hormone deficiency article, would you expect the promoter regions for GH, PRL and TSH to be identical, different, similar?
expect the promoter regions to be similar
In the melanoma reading, the oncogenes affect what?
the RAF/MEK/ERK (MAPK) pathway
In the melanoma reading, majority of melanomas have activating mutations in what?
BRAF
In the melanoma reading, what is the MAPK signaling axis pathway?
- extracellular -> growth factors
- RAS
- MAP3K -> A, B, & C RAFs
- MP2K -> MEK 1/2
- MAPK -> ERK 1/2
- phosphorylate transcription factors
In the melanoma reading, what is PLX4032 (BRAF inhibitor) known as?
VEMURAFENIB
What is the basis for hereditary persistence of fetal hemogloblin?
Fetal cells express different transactivators in development. The cells expressing the transactivator
would be earlier stage stem cells
What is the locus control region?
a regulatory element that enhances expression of linked genes
How do we switch from Fetal to adult Globin?
- Missing Fetal globin Transactivator (not expressed anymore)
- HDAC keeps fetal genes condensed
How can we deliver more oxygen to tissues?
HDAC inhibitor (vorinostat) to stop condensing of fetal hemoglobin gene
What drug was approved for treatment of refractory cutaneous T-cell lymphoma?
vorinostat
What is a treatment for sickle cell anemia?
HDAC inhibitor (vorinostat)
From the T-box article, what genes are mutated in cardiac congenital anomaly syndromes?
TBX1 & TBX5
From the T-box article, what happems when the TBX genes were removed from the mouse?
morphological differences in the mouse’s heart
An extracellular signal can lead to what?
- change in conformation of a receptor
- signal transduction to nucleus
What is an example of a signal transduction pathway, leading to the activation of a transactivator and transcription?
NF-kappaB
What are the steps of TNF alpha leading to transcription?
- TNF-Alpha binds to the receptor
- Leads to phosphorylation of IKK (kinase)
- IKK will phosphorylate NFkappaB (transactivator) that will translocate to the nucleus
- NFkappaB BINDS TO DNA in the nucleus and activates transcription
What are the steps of JAK/STAT1?
- A cytokine (IFN) will bind to the cytokine receptor
- Causes the two subunits of the receptor to come together
- This causes JAK to cross phosphorylate each other
- The JAKs will then phosphorylate the receptors
- This attracts STAT s (transactivators) that will dimerize
- Dimerization of STATS hides their phosphates
- Dimerized STATS translocate to the nucleus and increase transcription of target genes
In JAK/STAT1, what internalizes the phosphate?
dimerization
Where is the Hedgehog pathway constituitively active in?
basal cell carcinoma
What are the steps of the Hedgehog pathway?
- Hedgehog will interact with patched
- Patched cant inhibit smoothened
- Smoothen interacts with protease
- Protease cant cleave ci protein
- We now have a co activator (rather than repressor)
- Leads to transcription of pro proliferation genes
What is the trend if Patched is mutated and pathway remains constituitively active?
will lead to constant expression of Hedgehog target genes and likely oncogenesis
What is the trend if Ci is NOT active?
Ci keeps a binding site for a coactivator-HAT complex, allowing for transcription of pro S-phase genes
Does Patched interefere with smoothened?
no
What are the steps of the receptor tyrosine pathway?
- Two receptors come together and phosphorylate each other (own tyrosines kinase activity)
- This causes the GTP exchange (AKA switch from GDP to GTP);The inactive RAS is bound to lose GDP, RAS is activated when it binds to GTP
- When RAS gets activated it starts up the downstream signals like RAF, MER, ERK, aka phosphorylation of transactivator
What can lead to an increased growth of spermatocytes?
FGF mutations
What can FGF mutations lead to?
achondroplasia (form of dwarfism)
Where do FGF mutations occur?
- in spermatocyte development
- in chondrocytes (cartilage)
- signaling pathway even with no PIC
What cant peptide hormones diffuse through the lipid bilateral membrane?
needs EXTRAcellular receptor
How can steroid hormones diffuse through the lipid bilateral membrane?
INTRAcellular receptors
What is a transcriptome?
- collection of RNA’s from a given sample/cell type
- the representation of genes expressed in a given cell type
Does the presence of an mRNA transcript automatically imply the translation of the mRNA?
NO
What can represent a fingerprint of a cell type in transcriptional regulation?
transcriptome
What is an RNAseq file?
a transcriptome that can be obtained by converting all of the mRNA in a cell to cDNA using reverse transcriptase in a test tube, then sequencing
What are the qualitative & quantitative properties of RNAseq?
- Qualitative: can be done for each specific gene of interest
- Quantitative: proportionality is maintained
What leads ot a larger RNAseq value?
the more reads for a gene -> cbioportal.org
What are the steps of creating an RNAseq file to obtain a transcriptome?
- sequence all the cDNA fragments
- align the fragments to a reference genome
- count the fragments at each position.
What is LTB in a cluster software?
example of a fingerprint distinct b/w 2 cancers
What is the key in reading the clsuter software?
- GREEN= LITTLE EXPRESSION
- RED= A LOT OF EXPRESSION
- BLACK=No difference IN EXPRESSION
The cluster software is helpful in what type of fingerprinting?
tumor cell
Are MM and CLL the same or different blood cancers?
different
The articles regarding “21-gene expression assay” & “blood transcriptome” are examples of what?
clinical significance to transcriptomes
Does the chromatin remodeling complex use ATP?
YES
In the chromatin remodeling complex, what can SSDBPs & transactivators recognize?
DNA sequences that are still compacted in nucleosomes
What are the modification states & meaning of the histone code?
- acetylated -> gene expression & histone deposition
- methylated -> gene silencing/heterochromatin (condensed/closed)
- phosphorylated -> mitosis/meiosis
- phosphorylated/acetylated -> gene expression
What are the steps & proteins of heterochromatin silencing?
- HDAC (“Close down DNA” -> takes away acetyl groups)
- Histone methylase (methylate K-9 in this example)
- HP1 (facilitates formation of condensed chromatin- aka heterochromatin): HP1 can self associate: HP1 also recruits DNMTs
- DNMT
- MBP -> repeats process
Where is MBP, from the heterochromatin silencing process, defective in?
Rhett syndrome -> defective heterochromatin spreading
Heterochromatin silencing can be as large as what?
as large as an entire female X chromatin that gets inactivated
Where do DNMTs add methyls to?
to cytosine
How is heterochromatin passed?
from parent to daughter cell
Symmetry of what makes methymethylating both strands occur in ALMOST the same place?
CpG
What represents cervical carcinoma?
HeLa cell
How do we get a shutdown of X chromosome?
epigenetics
What are the steps of Xist during RNA spreading?
- Xist gene (found in chromatin) is transcribed
- Xist coats the X chromosomes that becomes inactive (this is done around the 200 cell stage in embryonic development)
- Xist function as a scaffold for histone methylase (which facilitate heterochromatin silencing)
What does Xist RNA bind?
histone & DNA methylases
What are the characteristics of epigenetics?
- heritable change in genome
- NOT a mutation
- refelects gene slicing
- occurs at small regions
As we develop, what is the trend of methylation?
increases & decreases back/forth
What is the trend of imprinting between mother & father?
- If inherited from the dad -> the gene stays methylated (silences)
- If inherited from the mom -> gets demethylated (we do not know why)
What can prevent disease in imprinting?
if dominant mutant allele is methylated
What is Prader-Willi syndrome?
- defect in imprinting
- results in chromosome 15 deletion & repression of other chromosome by methylation
- patietns a part of autism specturm
Where are response or regulatory elements located?
100-300 nucleotides upstream of TATA box
