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Summary Learning outcomes AMB worked out
- Instelling
- Radboud Universiteit Nijmegen (RU)
learning outcomes AMB
[Meer zien]Voorbeeld 3 van de 19 pagina's
Voorbeeld 3 van de 19 pagina's
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Voorbeeld van de inhoud
Learning outcomes AMBTranscriptional control of gene expression (L1-3)
Regulation of transcription in prokaryotes:
Gene structure: coding sequence, promoter, untranslated region
Coding sequence: In prokaryotes, RNA polymerase cannot read ATG start sequence and therefor
there is a promoter region.
Promoter: The promoter consists a transcriptional start site which consist of two parts; at -35 and -10
nucleotides before the start site. These two sites are recognized by Sigma factor.
Untranslated region: The 5' UTR is typically between 3 and 10 nucleotides long (while in eukaryotes it
can be 100 nucleotides long)
General principle of transcription regulation (bacteria and eukaryotes)
Transcription initiation is a rate limiting step in gene expression and therefor it is a key point for
regulation. There are four ways of transcription (due to regulation):
1. Normal transcription, when polymerase recognizes the promoter and transcription occur.
2. Less transcription, when it is expressed at a lower level, but still expressed (other sigma factors)
3. Silenced genes, because there is no sigma factor or there is a repressor protein
4. Genes with higher expression, this can happen when for instance polymerase got a helper.
Transcription initiation, elongation, and termination
During initiation, the polymerase must bind to the promoter sequence of duplex DNA. Then the
polymerase melts the DNA near the transcription start site, forming a bubble. Then polymerase
catalyses the phosphodiester linkage of two initial rats.
Then during elongation, the polymerase transcribes 3’ to 5’ down the template strand, melting the
DNA and adding rNTPs to the growing RNA.
Termination is started at the transcription stop site, the polymerase releases the completed RNA and
dissociate from the DNA.
Sigma factors
Sigma factor recognizes both the sequences in the promoter, and it binds with polymerase and
transcription will start. Sigma factor is a three-in-one protein, because in one way it will define where
transcription should start, the direction of transcription and also extent define the intensity of the
transcription. (So, start point, direction and intensity). Most of the genes are recognized by sigma
factor 70 (σ70). σs is for a stationary-phase gene and general stress responses.
Regulation of transcription (lac operon, one- and two-component regulatory system)
Lac operon: is a repressor and binds very close to the sigma factor 70 on the operator (CAP helps to
stabilize the polymerase on the promoter). When the lac repressor binds to the operator, the binding
of RNA polymerase is blocked. Lactose binds to the lac repressor and this leads to a conformational
change and no binding to DNA and no repression. cAMP can bind to the CAP protein leading to
binding to the DNA and activation of transcription.
One component regulatory system: The sensor and activator are in one complex (CAP protein) (all
function in one protein). This is a two-in-one protein because it has a domain which is specific to bind
a cAMP and another part of the protein is specific to bind to the DNA. When cAMP is present and
binds conformational change and this leads to extension of two helicases and change the
orientation and the protein can bind to DNA.
Two component regulatory system: The sensor and regulator are separated. NtrB (sensor protein)
senses Gln and NtrC (regulatory protein and interact with σ54 from a distance) binds to the DNA and
interacts with the polymerase to promote transcription.
High level of Gln, there is no need for expression of downstream genes, the kinase domain is inactive,
and the DNA binding domain cannot bind no transcription.
,Learning outcomes AMB
Low level of Gln, NtrB changes conformation and kinase domain become active and phosphorylates
NtrC changes conformation leading to binding DNA and resulting in transcription.
Differences in bacterial and eukaryotic transcription regulation
The prokaryotes have mainly polycitronic genes and the regulatory sequences are located near the
transcription start site. There is also 1 RNA polymerase with multiple sigma factors.
In eukaryotes the gene are regulated individually, and the regulatory sequences can be several kilo
bases apart. There are 3 RNA polymerases with separated transcription initiation complexes and
many eukaryotic genes contain introns and sometimes multiple promoters.
Regulation of transcription in eukaryotes:
RNA polymerase I, II, and II, and corresponding class I, II, and II genes
RNA polymerase I synthesizes pre-mRNA (28S, 18S, 5.8S) and localizes to the nucleus. It is most likely
to separated out because it gives rise to about 90-95% percent of the total RNA content of the cell.
RNA polymerase II synthesizes mRNA, small nuclear RNA (U1, U2, U4, and U5) involved in mRNA
splicing as well as miRNA. It is for the transcription of genes. Also, RNA polymerase has a specific
subunit called CTD (C-terminal domain).
RNA polymerase III synthesizes tRNA, 5S and &s rRNA and a couple of other small RNAs.
CTD (RNA polymerase II), role of CTD phosphorylation
The subunit 1 of the RNA polymerase II has a CTD (C-terminal domain). It is a repeat sequence and it
is a major place for regulating the activity of RNA polymerase II and is regulated by phosphorylation.
The phosphorylated CTD is involved in elongation (CTD-P is present in puffs and makes active
transcription). The unphosphorylated CTD is associated with little transcription. The CTD code is
complex, because the serine can be phosphorylated which is achieved by the activity of kinases. This
can be reversed, and it can be dephosphorylated by phosphatase which ‘erase’ the phosphorylation
mark. It serves as a signal for recruitment of enzyme complexes involved in numerous transcriptions
coupled processes.
Core promoter, promoter-proximal elements, enhancer
Core promoter elements: is surrounding the transcription start site (about -40 or +40 bp from the
start site) and is a minimal sequence required for the assembly of the preinitiation complex. The
TATA box, initiator, downstream promoter element (DPE) are the most common core promoter
elements and support different promoter recognition mechanisms.
Promoter-proximal elements: is upstream of the core promoter (-200 - -40 bp), will regulate the
activity of the polymerase and is often gene- or cell-specific.
Enhancer: is up-/downstream of the core promoter (further than 200 bp), will regulate the activity of
the polymerase, work from a distance, is orientation-independent, and is often gene- or cell-specific.
Core promoter elements: TATA box, initiator, DPE, CpG islets
Consist of the combination of distinct sequence motifs. The TATA box fulfils a similar function as the
binding site for the sigma factor. Mammalians do not have a core promoter element (TATA box) but
contain CpG islets. The CpG islets contain numerous CG nucleotides and represents a different
mechanism for promoter recognition.
Pre-initiation complex formation, sequential recruitment of general transcription factors (TFIIA, B, E,
F, H)
The preinitiation complex is the formation and binding of all the different general transcription
factors to the core promoter elements. The preinitiation complex is the general transcription factors
and RNA polymerase II. These general transcription factors are all required for polymerase II
transcriptional initiation. Activators and repressors can influence the efficiency of the preinitiation
complex (PIC) as well as the promoter clearance.
, Learning outcomes AMB
In vitro: TBP binds to the TATA box and bends the DNA. TBIIB binds downstream of the TBP and
stabilize the binding of TBP on the core promoter element. Then TFIIE binds and enables the binding
of TFIIH and TFIIH has two important functions. As helicase it uses ATP to pry apart the double helix
at the transcription start site allowing transcription to begin. As a kinase is will phosphorylate RNA
polymerase II, releasing it form the general factors so it can begin the elongation process of
transcription.
TBP, TFIID, TAFs
The binding of the PIC to the sequence was taught to be by the TATA box which is recognized by the
TATA Binding Protein (TBP). TBP has a number of TBP associated factors (TAFs) and together they
form the TFIID complex. This complex binds to the promotor via the TATA box. When there is no
TATA box present, TFIID binds via certain TAFs to the initiator sequence and the downstream
promoter element (DPE). Some do not even contain initiator sequences and DPE but contain CpG
islet. The GC box is bind by a protein called Sp1, and this can recruit the TFIID complex to the
promoter regions.
Holo-enzyme complex, mediator
In vivo some PIC components assemble to complexes prior to binding to the core promoter.
Polymerase II, TFIIF, TFIIE, TFIIH assemble together and form a holo-enzyme complex. The holo-
enzyme complex is not the PIC because the holo-enzyme complex does not contain TFIID, TFIIA,
TFIIB. The mediator complex is also a part of the holo-enzyme complex and is the bridge between the
RNA polymerase II and the activators and repressors.
Experimental analysis of regulatory elements (e.g. promoter deletion analysis, in vitro transcription
assay)
Promoter deletion analysis: You take a plasmid which contain an easily detectible proteins (GFP) and
on the other hand you take you DNA sequence and you start to truncate it. The transcription start
site has to be included because the core promoter has to be close by. You then delete small pieces
again and again on the 5’ until you reach a small fragment. Then you can generate multiple
constructs, you clone these DNA fragments into a vector and then you end up having a couple of
plasmid constructs. Then you transfect this into cultured cells and then you can look at the activity of
the reporter gene. Now you can determine where the gene regulator elements are. But it is hard to
determine sometimes if you delete a repressor or an activator.
In vitro transcription assay: instead of just truncated it at the 5’ end, you can delete certain areas
with a little bit of overlap. You then can figure out there is something different between two
constructs and then there has to be some regulatory element there. When you hit the core promoter
element the effect goes to zero.
Transcription factor structure (DNA binding, activation/repression domains)
Prokaryotes have a repressor which sometimes ‘sits’ on the promoter sequence or close by and the
polymerase cannot bind. Eukaryotes repressors might interact with the PIC from a distance, and they
also have a repressor domain.
They have to bind to DNA and recognize a specific DNA element (=DNA binding domain) and there
are many different DNA binding domains.
Activation/repression domains: they have to interact with the mediator or with the PIC. Often these
domains act trough other proteins, called co-activator/repressors. the activation domain of the
nuclear receptor proteins is more structures and undergo conformational changes upon ligand
binding.
Examples of DNA binding domains
Homeodomain proteins: Made of three helices, 1 of them is directly interaction with DNA. The other
two are used to position it in the right orientation.
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