Ferrari: Lecture VII Flashcards
Eukaryotic Gene Control
What makes the difference between different cells of the same organism?
the regulation of gene expression (how the transcriptome and the proteasome are regulated)
Are the decision taken at a molecular level during development and differentiation reversible?
not in complex organisms (only in bacteria)
What are the regions on the DNA that control gene expression?
promoter
regulatory sequences
regulatory proteins & transcription factors
interfering RNA
Promoter
essential part of the gene responsible for expression by binding the RNA polymerase with the accessory TF (true for polymerase I, II and II in eukaryotes)
Regulatory Sequences
allow the binding of the regulatory proteins
Regulatory Proteins and Transcription Factors
role in gene expression and regulation
(RNA can also control gene expression)
Interfering RNA
regulation of gene expression
How are things brought closer to the promoter?
looping of DNA (job of enhancers)
What are 2 main actors responsible for the looping of DNA?
trans-acting proteins
cis-acting elements
trans-acting proteins
in transcription factors
cis-acting elements
on the DNA; they are sequences recognized by the TF on DNA
How is transcription controlled?
binding of trans-acting proteins to the cis-acting regulatory DNA sequences
ENCODE Project
objective was to identify all the elements on the DNA and do all the annotations on them in a functional way
Sequences of Regulatory Proteins (cis-regions) act in genes that are usually involved in:
DNA replication
chromatin condensation
chromosome regulation
gene expression
What is the most used model of simple eukaryotes?
Yeast Saccharomyces Cerevisiae and Saccharomyces Probe
WHy are Yeast Saccharomyces Cerevisiae and Saccharomyces Probe the most used simple eukaryotic model?
the regulatory regions are very short, they are easy to study, and they are close to the core promoter
What was hard about studying complex eukaryotes?
the elements (enhancers, insulators, or silencers) were far away and hard to identify, but now we know the distance between promoter and regulatory sequences
Enhancers
cis-acting elements on the DNA which offer multiple binding sites for TF
What is the general size of enhancers?
500bp
What do enhancers do?
relay different regulatory signals that come from activators to the basal machinery of the promoter
Enhanceosome
soma of the enhancer that defines all the TF that are bound to the same enhancer (needed because there are multiple binding sites for different proteins on the enhancer)
What is an example of the enhanceosome?
beta-interferon gene
In the beta-interferon complex, what is the function of HMG1?
bends the DNA and allows for the interaction between proteins and DNA at different distances
How many TF are there in humans?
more than 3000
What are the 3 classes of regulatory proteins?
sequence-specific DNA-binding proteins
general transcription factors (GTFs)
chromatin remodeling and modification complex
general transcription factors (GTFs)
recognize the same sequence in all the genes in the promoters
part of the RNA polymerase II complex
required for promoter recognition and catalysis of RNA synthesis
chromatin remodeling and modification complex
change chromatin by assisting the transcriptional machinery in order to facilitate access of proteins to DNA
RNA polymerase II
initiates transcription without TF because it has affinity for the DNA, but without the TF, it cannot bind in a specific way
What do transcription factors do for RNA polymerase II?
bring specific polymerase to a specific region
RNA polymerase II promoter
region upstream of the coding region for the +1 (+1 is where transcription starts and the promoter is usually 200bp upstream
What defines a core promoter?
core promoter is the minimal sequence needed for effective transcription
Which sequences identify the presence of a promoter?
TATA BOX
BRE
INITIATOR
DCE & DPE
TATA BOX
fixed position in prokaryotes and eukaryotes (all transcription starts at +1, and all upstream sequences are annotated by - and all downstream are annotated by +)
TATAAT is the TATA binding protein sequence (TBP sequence)
BRE
sequence rich in G & C
binding site for TF2B, a general transcription factor of RNA polymerase II
it is the TF2B recognition element
INITIATOR
middle of the sequence and includes the +1 (so it is not upstream like the TATA BOX)
it has a couple of nucleotides upstream and 4 nucleotides downstream the +1
DCE & DPE
sequences are all bound to TF2D and works with RNA polymerase II
Why are the TATA BOX or the other listed sequences needed?
they are needed so that the DNA can bend and bind
How are RNA polymerases recruited on promoters?
by TBP containing complexes
role of polymerase I
enzyme that transcribes for ribosomal RNA
role of polymerase III
transcribes all the transfer RNA and 5S ribosomal RNA
What TFs is polymerase II associated with?
TF2E
TF2F
Tf2H (helicase)
Why is TF2H (helicase) needed?
needed to open the double stranded DNA to start transcription
When are the factors that facilitate transcription released?
once they enter the elongation phase
What are the major changes in polymerase II when transcription is in the elongation phase?
phosphorylation in the C-terminal domain, which is needed to change kinetics from transcription initiation to transcription elongation
Polymerase II is also a binding site for other factors required during transcription…what are these factors?
splicing factors as they all occur on the C-terminus of the enzyme
What 2 things happen cotranscriptionally at the C-terminus of enzymes? Why?
phosphorylation to change transcription kinetics
splicing of RNA
makes the process more efficient
Cofactors
large complexes that mediate interactions between regulatory factors and general transcription factors using activators or repressors
Mediator
mediates the interaction between transcription factors (activators) and RNA polymerase II
Analyze the gene regulation graph of eukaryotes:
How can regulatory proteins bind DNA?
they use transcription factors to identify the pattern of particular chemical groups, such as:
hydrogen bond donors
hydrogen bond acceptors
hydrophobic patches
Where does TF recognition occur?
ONLY in major grooves as the patterns are marked differently here
What kind of features must regulatory proteins have?
DNA binding domain
activation domain
flexible domain
DNA binding domain
responsible for the interaction with the DNA (can be present in the N-ter/C-ter/middle of the protein)
activation domain
interacts with other proteins
flexible domain
links together other domains
DNA-binding domain has different motifs. Every TF must have one of these motifs:
Helix-turn-helix
Homeodomain
Zinc-finger
Winged helix (forkhead)
Leucine-zipper
Helix-loop-helix
Helix-turn-helix (simplest motif)
characterized by an 𝛼-helix (blue), a small turn (white), and another 𝛼-helix (red)
Zinc-finger
zinc atom linked to a cysteine or histidine residue
the zinc atom is linked to four amino acids: they can be cysteines or histidines in any combination - e.g., 4xHis, 3xHis and 1xCys, 2xHis and 2xCys…
generally, in TF there is more than 1 zinc-finger domain
Leucin-Zipper
leucine frequency is high
form homodimers or heterodimers, which can alter the DNA-binding ability of the TF and a way to expand regulation
What is heterodimerization an example of?
combinatorial control (combination of different proteins control a cellular process, which increases the number of DNA sites that are recognized)
What is special about the N-terminal tail of histones?
protrude outside the nucleosome and can be modified post-translationally by enzymes that reside in the nucleus
Describe the histone modification: acetylation of lysines:
removes the charge from the lysine and promotes the formation of euchromatin (less compact chromatin) and allows transcription of nearby genes
deacetylation: repression
hyperacetylation: activation
What is the most famous acetyltransferase in eukaryotes?
p300/CBP complex
What does the p300/CBP complex do?
interacts with activators and is responsible for the acetylation of histone 4
What does the acetyltransferase, PCAF, do?
preferentially acetylates histone 3
Acetylation is always related to __________
enhanced expression
Histone methylation can have different outcomes based on:
_____
_____
_____
number of methyl groups
residue
histone that is methylated
The region in a genome that is transcriptionally active is in the presence of _____
euchromatin
The region in a genome that is transcriptionally repressed is in the presence of _____
heterochromatin
Why do we need factors that remodel nucleosomes?
because the position of adjacent nucleosomes varies a lot
What are chromatin remodelling complexes?
found in eukaryotic cells
use energy of ATP hydrolysis to change the structure of nucleosomes temporarily so that DNA becomes less tightly bound to the histone core
What does the remodelling of nucleosomes permit?
access to nucleosomal DNA by proteins involved in several processes such as gene expression, DNA replication and repair
What is the most famous chromatin remodelling complex? Why?
SWI/SNF family
it is a complex of proteins that is very conserved among different species
What is conserved in the SWI/SNF family?
presence of an ATPase domain, which requires energy to remodel the chromatin
During the transcription of RNA, there is the cycling mechanism of nucleosome remodelling. When is this disrupted?
ONLY during DNA replication
ATP-dependent remodelling complexes….
loosen the interaction between DNA and histones to permit the entrance of DNA-binding proteins
Once DNA-binding proteins have done their job, another ATP-dependent remodelling complex….
restores the normal nucleosome structure
Histone-modifying enzymes and chromatin remodelling enzymes…
work together to release and recondense stretches of chromatin
Where does DNA bending occur?
minor groove of double helix
