Lesson 7 Flashcards
what creates the difference between cells in the same organism?
the regulation of gene expression → how the transcriptome and the proteome is regulated
what is the function of the promotor region?
essential part of the gene → region that allows the binding of the RNA polymerase with all the accessory transcription factors
what are regulatory sequences?
sequences on the DNA which allow the binding of the regulatory proteins ; not usually part of the promoter in terms of spatial occupancy, but they also can be very far from the basal promoter
what can control gene expression besides regulatory proteins and transcription factors?
RNA
what types of RNA can regulate gene expression besides protein transcription factors?
interferring RNA and regulatory RNA
what are the two main actors in regulation of the genome in complex eukaryotes?
trans-acting proteins and cis-acting proteins
what are the trans-acting proteins?
transcription factors
what serve as the cis-acting proteins?
sequences recognized by the transcription factor on the DNA (also on the DNA themselves)
what is transcription ultimately controlled by in complex eukaryotes?
the binding of trans-acting proteins to cis-acting regulatory DNA sequences
what was the goal of the ENCODE project?
to identify all the elements on the DNA and do all the annotations on them in a functional way in order to discover their functions
cis-regions usually act in genes that are involved in what four activities?
- DNA replication
- chromatin condensation
- chromosome segregation
- gene expression
describe the regulatory regions in simple eukaryotes?
very short sequences that are easy to study as they are close to the core and the basal promotor
why is it more difficult to study enhancers, insulators, and silencers in complex eukaryotes?
these elements are very interspersed in the genomic DNA and can be very far from each other
when distal elements like enhancers or silencers are far away from each other, what must occur?
the interactions with elements on the promotor must be mediated by other proteins (must have something to mediate the cross-talking between elements)
what are the main actors of regulation on the DNA?
enhancers
what are enhancers classified as?
cis-acting elements
what are enhancers defined as?
sequences on the DNA which offer multiple binding sites for transcription factors
where is the enhanceosome usually located and what does it define?
located in the soma of the enhancer and defines all the transcription factors that are bound to the same enhancer (it had multiple blinding sites for transcription factors)
during evolution, what has increased complexity?
an increase in the number of transcription factors → increase in regulation increases complexity
what three categories do proteins regulating transcription fall into?
sequence-specific DNA binding proteins, general transcription factors (GTFs), and chromatin remodeling and modification complexes
describe sequence-specific DNA binding proteins:
they should have a DNA binding domain and they recognize specific bases on DNA
what two groups are sequence-specific DNA binding proteins divided into?
activators and repressors
what is the function of general transcription factors?
recognize the same sequence in all genes
what larger complex are GTFs part of?
RNA polymerase II → recognize a sequence that is the same in all promotors
what is the function of chromatin remodeling and modification complexes?
changing chromatin by assisting the transcriptional machinery in order to facilitate the access of proteins to DNA → regulatory proteins need to see the sequence for the binding and these complexes are able to facilitate the reading of the DNA.
Enzymes for the synthesis of RNA, in particular RNA pol 2 (that transcribes for mRNA) can’t initiate transcription without the general transcription factors, why not?
RNA polymerase has some affinity for DNA, but without them it cannot bind in a specific way → TFs bring the specific polymerase to the specific region (otherwise initiation is not specific)
where does the +1 region refer to?
where transcription starts and promoter is usually 200 bp upstream
what does RNA pol 2 control?
regulation of messenger RNA
where is a promotor located?
region of the upstream the coding region for the +1
what is the core promotor?
the minimal sequence needed for effective transcription and in order to identify the promotor
describe the position of the TATA box:
fixed position in eukaryotes and prokaryotes from the transcriptional start sites
how are upstream sequences connotated?
-
how are downstream sequences connotated?
+
what is the TATA box?
the TATA binding protein sequence
what has a specific sequence rich in G and C, that is also the binding site for TF2B (general transcription factor for pol2)?
BRE
what is BRE?
the TF2B recognition element
what are two essential components of the promotor?
TATA Box and BRE
besides the TATA Box and BRE, what else can be present in eukaryotes?
an initiator and the DCE / DPE
where is the initiator located?
it is in the middle of the sequence and includes the +1 (so is not upstream
sequence like the TATA box) → has a couple of nucleotides upstream and 4 nucleotides downstream the +1
what are DCE and DPE bound to?
TF2D
what is TF2D?
one of the other general transcription factors working with RNA pol 2
what is a characteristic of the TATA box that allows the probability to find this specific sequence of bases in this exact position very high?
it is a consensus sequence
if a gene in the human genome does not have a TATA box, what other combinations might serve as the promotor?
DPE + the initiation factor or an initiator + a CPG island (sequence made of repeating C and G in the promotor)
we need the TATA box in order to bind TBP (TATA binding protein), which has what function?
to bind DNA
once TBP binds to the Tata box, what occurs?
the bending of DNA
where does DNA bending occur?
at the level of the minor groove of the double helix
what is the purpose of DNA bending?
works like a marker on the chromatin → like a flag for all the other transcription factors to mark the promotor gene
what is the function of TBP?
recruits RNA polymerase and tells it where to bind = essential protein
what is the role of RNA polymerase I?
enzyme that transcribes ribosomal RNA
what is the role of RNA polymerase III?
transcribes all tRNA and 5S rRNA
what are the three rRNA in eukaryotes?
5S, 18S, and 28S
what are the smallest RNA?
tRNA
what makes the genes encoding for ribosomal RNA unique?
they are some of the few genes in the eukaryotic genome which have repeated sequences in tandem
in the first step of transcription, what needs to bind to RNA polymerase II?
we need the binding of other proteins such as TBP but also TF2B that binds to BRE in order to let pol 2 reach the promotor
besides TF2B, what other transcription factors is RNA poly 2 associated with?
TF2F, TF2E, and TF2H
what is TF2H and why is it important?
it is a helices → needed to open the double stranded of DNA to start transcription
once transcription is initiated and enters the elongation phase, what occurs?
all factors that facilitate transcription are released because they are no longer needed
what is the major change on pol II when transcription enters the elongation phase?
phosphorylation of the C-term domain is needed in order to change the kinetics from transcription initiation to transcription elongation
what happens simultaneously as pol II moves along the DNA transcribing mRNA?
splicing also happens cotranscriptionally
where are all factors required for the splicing of RNA associated?
C-term of the enzyme → so that splicing occurs co-transcriptionally in order to make the process very efficient
what are cofactors?
large complexes that interact with either activators or repressors of transcription and RNA pol II to enhance or repress transcription
what is a mediator?
one big complex made by more than 25 subunits, and mediates the interaction between transcription factors (activators) and RNA polymerase II
what is the main function of mediator complexes?
transmit signals from the transcription factors to the polymerase, acting as a big co-activator in all eukaryotes
where have mediator complexes been found?
both simple (yeast) and complex (human) eukaryotes
what must happen in order to facilitate the the interaction between proteins near the transcription start site (TSS) and proteins that might be bound to distal DNA sequences?
DNA bending
what is the the complex mediating this protein-to-protein interaction and that conveys the signal from activators to RNA pol II in order to promote bending?
mediator
what must transcription factors be able to recognize on the outer part of the double helix?
the pattern of particular chemical groups
where do transcription factors recognize the pattern of particular chemical groups on the outer part of the double helix?
only in major grooves
why do transcription factors only recognize the pattern in major grooves?
only in major grooves are the patterns marked different for each of the four base pair arrangements
what also determines the overall geometry of the double helix by creating distortions of the helix that can be recognized by proteins?
nucleotide sequences
what three features must regulatory proteins have?
a DNA binding domain, an active domain, and a flexible domain
describe the dna binding domain of a regulatory protein:
can be present in the N-ter/C-ter/middle part of the protein and is responsible for the interaction with the DNA
what is the function of the active domain in regulatory proteins?
interacts with other proteins
what is the function of a flexible domain in regulatory proteins?
links together other domains
dna-binding domains can be structured in different motifs, however, every TF must have one of which motifs?
- Helix-turn-helix
- Homeodomain
- Zinc-finger
- Winged helix (forkhead)
- Leucine-zipper
- Helix-loop-helix
what is the simplest DNA binding motif, and how is it structured?
helix-turn-helix → presence of an alpha helix, a small turn, and another alpha helix
what is a prominent dna binding motif widely used in molecular engineering, and what is its structure?
zinc-finger → a zinc atom linked to a cysteine or histidine residue
what is another important dna binding motif?
leucine-zipper → high leucine frequency, form homodimers or heterodimers: this can alter the specificity of the DNA-binding ability of the transcription factor and is also a way to expand the regulation
what are nucleosomes composed of?
DNA wrapped around a protein complex (usually a histone)
because the N-terminal tail of histones protrudes outside the nucleosome, how can it be modified?
post-transcriptionally
how are nucleosome modifications added and removed?
enzymes
what is the most well-known nucleosome modification?
acetylation of lysines
what happens during the acetylation of lysines?
removes the charge from lysine thus promoting the formation of euchromatin and allowing the transcription of nearby genes
what is hyperacetylation directly related to?
activation
what is de-acetylation related to?
repression
what is one of the most famous acetyltransferases?
p300/CBP complex
what is the function of the p300 CBP complex?
responsible for the acetylation on histone 4
what is the function of the acetyltransferase protein PCAF?
preferentially acetylates histone 3
what is acetylation ALWAYS related to?
enhanced expression
describe the status of chromatin in the presence of euchromatin:
transcriptionally active
describe the status of chromatin in the presence of heterochromatin:
transcriptionally repressed
why do we need actors that remodel nucleosomes?
the positions of adjacent nucleosomes vary a lot
what use the energy of ATP hydrolysis to change the structure of nucleosomes temporarily so that DNA becomes less tightly bound to the histone core?
chromatin remodeling complexes
what is the most famous chromatin remodeling complex family?
SWI/SNF family → very much conserved among different species
what do the SWI/SNF families of different species have in common?
presence of an ATPase domain
when is the only time that we need to disrupt the nucleosomes structure when transcribing RNA?
during dna replication
what is the function of ATP-dependent remodeling complexes?
loosen the interaction between dna and histones thus permitting the entrance of dna binding proteins
after dna binding proteins have done their job, what occurs?
another dna binding protein restores the normal nucleosome structure
both histone-modifying enzymes and chromatin remodeling complexes work together to do what?
release and recondenes stretches of chromatin
