Unit 9: Eukaryotic Transcription Flashcards
What are the differences between prokaryotic and eukaryotic transcription?
1) bacterial transcription occurs on a DNA template and in eukaryotes transcription occurs on chromatin
2) in prokaryotes RNA pol can scan the DNA to find and bind to the promoter. Inn Eukaryotes RNA polymerase cannot read the DNA and instead uses transcription factors that must bind to the DNA at specific sites before the RNA polymerase can bind
What is chromatin? What is chromatins main component?
-The DNA in eukaryotes that is associated w/proteins. this DNA + protein form chromatin and the primary components of chromatic are histones
What are histones?
-proteins that compact the DNA
What must happen to the chromatin in eukaryotes in order for the RNA polymerase to bind to the promoter?
-the chromatin must be opened
Why are transcription factors needed for eukaryotic transcription but not prokaryotic?
-b/c prokaryotic RNAs cannot read the DNA w/o transcription factors
What do the basal transcription factors form? Why are these transcription factors needed?
-they form the initiation complex at the promoter region. These factors are needed for initiation and bind to DNA, allowing RNA polymerase to bind to the transcription factors of DNA complex with the core promoter
TRUE OR FALSE: Elongation in eukaryotes always need basal transcription factors
FALSE: these factors are needed for initiation but most are not needed for elongation
What is a core promoter?
-The shortest sequence at which an RNA polymerase can initiate transcription
What is the core promoter for RNA polymerase II?
-it is the minimal sequence at which the basal transcription apparatus can assemble, and it includes three sequence elements: The Inr, the TATA box, and the DPE (downstream promoter element)
What are the three sequence elements of the core promoter?
1) the Inr (initiator)
2) the TATA box
3) the DPE (downstream promoter element)
What forms the basal transcription apparatus?
-the basal factors (initiation complex) joined to the RNA polymerase forms the basal transcription apparatus.
What is an enhacer?
-a cis-acting sequence that increases the utilization of most eukaryotic promoters and can function in either orientation and in any location (up or downstream) relative to the promoter
what is a silencer?
-a short sequence of DNA that can inactivate expression of genes in its vicinity (this is an example of negative regulation)
What are housekeeping genes?
genes that are constitutively expressed in all cells b/c they provide basic functions needed for sustenance/survival of all cell types
What is a coactivator?
-factors required for transcription that do not bind DNA, but are required for DNA-binding activators to interact w/the basal transcription factos
What does RNA polymerase I synthesize?
-RNA pol I synthesizes ribosomal RNA (rRNA) in the nucleolus
What does RNA polymerase II synthesize?
- it synthesizes messenger RNA (mRNA) in the nucleoplasm and a few small RNAs
- it is also responsible for heterogenous nuclear RNA (hnRNA), which is a precursor for most of the mRNA
What is heterogenous nuclear RNA (hnRNA)?
-RNA that consists of transcripts of nuclear genes made primarily by RNA pol II; it has a wide size distribution and variable stability
What does polymerase III synthesize?
- it transcribes transfer RNA (tRNA), ribosomal RNA (rRNA), and some small RNAs in the nucleoplasm
TRUE OR FALSE: Some subunits are common to all three eukaryotic RNA polymerase?
TRUE; like TBP (TATA binding protein)
WHat is the largest subunit in RNA polymerase II?
-Carboxy-terminal domain or CTD, which consists of multiple repeats of a heptamer/heptapetide (multiple consesus sequence of seven amino acids)
TRUE OR FALSE: The two largest subunits in eukaryotic polymerase are homologous to the beta and beta prime subunit of the bacteria RNA polymerase
TRUE
Is there a homologous subunit in eukaryotic RNA polymerase that is similar to the sigma factor in prokaryotes?
-no b/c the function of the sigma factor is taken over by the basal transcription factors
What is CTD? What is the role of CTD?
- carboxy terminal domain that consists of multiple repeats of a consensus sequence of 7 amino acids or heptapeptides (deletion of more than half of these repeats can be lethal)
- CTD is phosphorylated at several serine and threonine residues and it is involved in regulating the initiation process, elongation, mRNA processing, and transport of mRNA into the cytoplasm.
RNA polymerase I has a single promoter that consists of two regions. What are these regions and what is their role?
1) the core promoter
2) UPE - Upstream Promoter Element (bipartite promoter)
What is the role of UBF1?
-a factor that wraps DNA around a protein structure to bring the core promoter and the UPE close to each other in RNA pol I
what are nontranscribed spacers?
The region between transcription units in a tandem (in sequence) gene cluster
In RNA polymerase I there are several transcription units w/each transcription unit coding for the large 28S and small 18S ribosomal RNA
What forms the holoenzyme in eukaryotes?
-RNA pol I + additional factors that are needed for initiation form the holoenzyme
additional factors: SL1 (TBP + TF2B) and UBF
How does RNA polymerase I recognize the promoter?
1) SL1
2) UBF
RNA polymerase I binds to the UBF-SL1 complex at the core promoter
What is SL1?
- SL1 is a transcription factor needed by RNA pol I to bind to the core promoter.
- SL1 binds to the core promoter (it binds before UBF) and its function is to make sure that the RNA polymerases properly localize at the start point, as well as for promoter escape. SL1 on its own enables low basal frequency and needs UBF for high frequencies
SL1 has two components: TATA BINDING PROTEIN (TBP) and TF2B factor of RNA pol 2
What is UBF? and where does it bind?
- also a transcription factor needed by RNA Pol 1 in combo with SL1 for high frequency initiation
- UBF has several functions including maintaining the open chromatin structure, stimulating promoter release, and stimulating SL1
- UBF binds to GC rich regions in the UPE and also binds to the minor groove of DNA and wraps the DNA around the protein bringing the core and UPE in close proximity
What is TBP?
- TATA binding protein
- Its role is to position the RNA polymerase correctly at the start site and is required for initiation of all RNA pol I, II, and III
TRUE OR FALSE: SL1 by itself enables initiation at high basal frequencies?
FALSE: SL1 by itself enables initiation at a low basal frequency. initiating at high frequency involves the second factor UBF.
What does RNA polymerase I require for efficient elongation?
special factor called Paf1 complex
RNA pol III has two types of promoters that fall intto three classes. What are the three classes?
Internal promoters type 1 and type 2
Type 1: box A and Box C , so TFIIIA binds to box A and TFIIIC binds to C and this recruits TFIIIB (TFiiiB contains the TBP) — type one is seen only in 5S rRNA
Type 2: Box A and Box B, TFiiiC binds to both box A and B, which recruits TFiiiB, which recruits RNA pol III to start site
Internal promoters cause initiation to occur where? Where is the promoter located?
-Internal promoters have short consensus sequences located w/in the transcription unit and cause initiation to occur at a fixed distance UPSTREAM
the internal promoter is located DOWNSTREAM
What are upstream promoters?
- class 3 promoters that lie upstream of the start site
- contain 3 short consensus sequences upstream of the the start site that are bound by transcription factors.
Irrespective of what class the promoter is, what element is necessary for promoter function?
-the element that is necessary for promoter function is a specific DNA sequence that is recognized by transcription factors which in turn recruits RNA polymerase to the promoter.
What does it mean when a promoter is bipartite?
this means that the promoters have two short sequence elements that are separated by a variable sequence.
EXAMPLE: figure 20.4
Type 1 promoter has box A separated from box C by intermediate IE, while Type 2 Box A is separated from Box B.
What are assembly factors?
-proteins that are required for formation of a macromolecular structure but are not themselves part of the structure
example TF3A and TF3C, which bind to the consensus sequences and enable TF3B to bind at the start point
Describe how TF3A , TF3B, and TF3C play a role in type 2 promoter and recruitment of RNA pol III?
1) In type 2 the TF3C binds to both box A and box B
2) it recruits TF3B which in turn recruits the RNA polymerase 3.
once TF3B is bound TF3A and C can be released without affecting the initiation reaction
Type III initiation occurs where?
-occurs upstream of the start site that contains the TATA box
What is the preinitiation complex?
-the assembly of transcription factors at the promoter before RNA polymerase binds in eukaryotic transcription
What are the three subunits of TF3B?
1) TBP - positions the RNA pol correctly at start site.
2) BRF (similar to TF2B used by RNA pol 2 and to SL1)
3) B99 - which initiates the transcription bubble
* after recruitment near the start side, its presence is sufficient to allow the polymerase to recognize and bind at the start site
What role does the TATA element play in Type III promoter?
- TF3B binds directly to the region including the TATA box for upstream promoters or type 3 promoters
What is the difference in the binding of TF3B, in type I, Type II, and Type III promoters ?
in type one and type 2 promoters TF3B binds just upstream of the start side while in type 3 or the upstream promoters the TF3B binds directly to the region including the TATA box
Which transcription factors are required by RNA polymerase II at all promoters?
-basal transcription factors are required by RNA pol II at all promoters - these factors are referred to as TF2X, where X is a letter that identifies the transcription factor
What are activators required for?
-activators are required to boost the efficiency of the core promoter (shortest sequence required that would enable transcription factors to assemble at the start site). The core promoter has low efficiency.
What is the Inr?
- The initiator region which is between -3 and +5 and is in the A with pyrimidines on either side (Py2CAPy5), where p are pyrimidines
The Tata box is surrounded by what type of sequences?
GC rich sequences;This is similar to the -10 box and bacterial promoters and located about 25 base pairs upstream the start site
What are TATA less promoters?
promoters that do not contain the TATA element or call the TATA less promoters. these promoters typically contain another element DPE or downstream promoter element which is located 28 to 32 residues downstream of the start site
What is the downstream promoter element (DPE)?
-is a common component of RNA polymerase II promoters that do not contain a TATA box (TATA-less promoters)
thus, rna pol 2 promoters either contain Inr + DPE or Inr + TATA box
What does TBP stand for and why is this name misleading? Which polymerases use TBP?
-Stands for TATA binding protein and its name is misleading b/c this protein was later found to be part of RNA pol I SL1 and RNA pol III TF3B, where there is no TATA element. Even in TATA less promoters, TBP is still required
What is the positioning factor for RNA Pol II?
-TF2D, which consists of 14 TBP associated factors or TAFs - some TAFs are tissue specific
How does TBP bind to the TATA box?
TBP binds to the minor groove in the DNA This is unusual as most DNA binding proteins bind to the major groove. In fact the crystal structure shows that it forms a sort of saddle structure over the minor groove of the DNA ,w/ the DNA being bent about 80 degrees.
This bending may corresponds energetically to the undwinding of DNA. In addition to spatial organization of the DNA on either side of the TATA Box is changed, allowing the transcription factors of RNA poly to be more closely associated w/the DNA
TAFs that resemble histones are found where?
in protein complexes that modify chromatin structure before transcription
What are the three types of chromatin?
1) The inactive gene enclosed chromatin
2) Potentially active gene in open chromatin (called poised genes)
3) A gene being turned on in an open chromatin
What is a poised gene?
an active gene in open chromatin; in poised gene the basal apparatus is all set up but it will not start transcription unless there’s a second signal. heat shocked genes are poised genes. they get activated immediately following a rise in temperature.
What are heat shocked genes?
-poised genes that get activated immediately following a rise in temperature
What is the first step in activating a TATA box containing a promoter in open chromatin?
-Binding of the TBP subunit of TF2D to the TATA Box
What needs to occur before the elongation step can proceed?
-RNA polymerase needs to be released from the promoter. This is called promoter clearance and is controlled by enhancers
What is promoter clearance and why is this step so important ? What is this step controlled by?
-releasing RNA polymerase from the promoter so that elongation step can proceed. This step is important b/c it determines if a poised gene or an active gene will be transcribed
What elements are required to melt DNA and allow movement of the polymerase?
TF2B
TF2E
TF2H
What occurs once TF2D is bound to the promoter site?
other transcription factors follow in a defined order. The large pre-initiation complex that is formed by the binding of different transcription factors extends the length of the protected region of the DNA
What does TF2D do when bound to a promoter/DNA?
TF2D. bound to DNA is in contact with the RNA polymerase near the site where the RNA exits. Here, it can play a role in abortive initiation or promoter escape.
How do transcriptions factors that bind enhancers bind the elements in the promoter?
They do not bind the elements in the promoter directly, rather they bind to another protein called a coactivator.
What is the common coactivator in promoter clearance?
Mediator
How do poised and active genes require that the transcription factors bound to enhancers interact with the promoter?
through DNA looping and the mediator
What are the last two promoters that join the initiation complex?
TF2E
TF2H
-they also act on the later stage of the initiation step
What is unique about TF2H?
TF2H is an important transcription fact that has several functions.
1) It acts as an ATPase, a helicase, and a kinase, which phosphorylates serine 5 on the C terminal domain of RNA polymerase.
2) It is also implicated in elongation as its interaction with DNA downstream the start point is required for the RNA polymerase to clear the promoter.
3) plays a role in DNA repair.
What are the steps In promoter clearance?
1) TF2B, TF2E and TF2H melt DNA to allow the movement of the polymerase
2) Phosphorylation of serine 5 and further phosphorylation of the CDT is needed at some promoters to end pausing and abortive initiation.
3) Further phosphorylation of CTD is done by the PTEFb complex
4) once the RNA polymerase escapes the promoter the initiation factors are released and the polymerases are not in elongation mode.
The transcription complex now consists of RNA pol II, basal transcription factors, TF2E, TF2H and all the factors and enzymes bound to the C-terminal domain of the polymerase
What is the PTEFb complex?
This complex phosphorylates CTD to promote promoter clearance. This complex is a CDK9 kinase and phosphorylation occurs on the serine 2 of the CTD heptapeptide repeat.
Explain the phosphorylation/modification of the C terminal domain of RNA polymerase 2 during transcription
1) When RNA polymerases is recruited to the pre-initiation complex the c-terminal domain is unphosphorylated
2) TF2H (part of the initiation complex) phosphorylates serine 5 which helps recruit the enzymes needed for capping the 5’ end of the transcript (guanylyl transferase is the enzyme)
3) guanylyl transferase binds to phosphorylated CTD and adds a G residue to the 5’ ends of the nascent RNA
4) PTEFb phosphorylates serine 2, which leads to the recruitment of proteins called scaff that in turn binds to splicing facts
How does eukaryotic transcription deal with genes that are several megabases long?
transcription of these would require transient histone modification or even disassembly and then reassembly.
What is needed to transcribe the chromatin through the nucleososmes?
TF2H
TF2S
1) each polymerase uses a chromatin remodeling complex that removes the histone H2A and H2B dimer from the octamer converting it to a hexamer which may be easier to displace.
When DNA damage occurs in which genes are preferentially repaired?
Transcribed genes
How does TF2H contribute to the repair process of damaged DNA?
TF2H has two subunits XPD and XPT that help in the creating of the transcription bubble and melt DNA at the sight of damage.
TRUE OR FALSE: An enhancer activates the promoter nearest to itself and can be any distance either upstream or downstream of the promoter
TRUE: yes they function through short range and long range interactions and can be up/downstream or even w/in a gene. and its sequence can be inverted w/respect to the promoter.
What is an activator?
a transcription factor that stimulates transcription
How do we tell the difference between and enhancer and silencer?
IF an element has more activators binding it then a repressor the it is an enhancer
IF more repressors than activators are binding then its a silencer
What are Upstream Activating Sequences (UAS)?
elements that are analogous to the enhancer
in yeast UAS behaves like an enhancer but works only upstream of the promoter and now downstream. They have a regulatory role
How does an enhancer, which can be some distance away from the promoter increase its activity?
- it is believed to do that by bringing proteins that are needed into the vicinity of the promoter in cis. Enhancers usually work only in cis configuration w/a target promoter
- The principle is that an enhance works in any situation in which it is constrained to be in the same proximity as the promoter
What happens if the enhancer and promoter are on different circular DNA?
-they do not interact
What happens to the enhancer and promoter if the DNA are catenated?
-they will function together
TRUE OR FALSE: Enhancers tend to act upon the nearest promoter
TRUE: although they can be distances away from promoter they will act upon the nearest
Why do we see DNA loops when looking at enhancers and promoters elements?
-if enhancer is some distance away from the promoter this would require the DNA to fold so that the enhancer and promoter elements, are in close proximity. This creates DNA loops.
TRUE OR FALSE: Gene expression is associated w/demethylation
TRUE: methylation is one of the forms of regulation promoter activity
What happens when a promoter is methylated?
- methylation is one of the forms of regulation promoter activity. Methylation of the promoter prevents transcription.
- this means that the residues have to be demethylated in order for the promoter to be active.
Where does methylation at promoters occur for RNA pol II?
-methylation at promoters for RNA pol II occur at the 5’ positions of C in a CG doublet by DNA methyltrasnferase
What are CpGs?
-CG doublets are referred to as CpG, which represents the C and G being on the same strand adjacent to each other, with the phosphate bond separating the two (different than CG base pairs)
Demethylation of the 5’ ends is necessary for what?
-it is necessary for transcription
What is one way to look for the distribution of methyl groups?
-using restriction enzymes that cleave the same target sequence but have different responses to the methylated state.
EXAMPLE: restriction endonucleases MSP1 and HPA2
- they both cleave CCGG sequences
- MSP1 cleaves irrespective of methylation, while HPA2 cleaves only non-methylated sites.
What are CpG islands and where do we see them?
- CpG islands are where we see a high density of dinucleotide sequence CpG.
- In some cases the CpG islands begin upstream of the promoter and extend downstream into the transcription unit. These islands surround the promoteres of constitutively expressed genes where they are unmethylated. CpG islands are also found at the promoters of some tissue-regulated genes.
in the structure of chromatin, what changes are associated w/gene expression?
- these changes include reduced content of H1 so the structure is likely to be less compact.
- other histones are acetylated, which is also a sign of gene expression.
- or there are sites which are empty of histone optimals.
TRUE OR FALSE: House keeping genes which are constitutively expressed have CpG islands
-TRUE, this accounts for about half of the islands. The remaining are found at promoters of tissue regulator genes. (in these genes though the CpG islands are unmethylated irrespective of the state of expression of the gene.
What are the two ways that methylation of the CpG island prevent transcription?
-methylation of the binding site for some factor may prevent its binding. This happens in the case of binding to a regulatory site other than the promoter
and methylation may cause specific repressors to bind to DNA
How is repression caused?
- repression is caused by either of the following types of proteins that bind to methylated GpC islands
1) The first type requires the presence of several methylated Cpg Doublets (MeCP1 is an example of this)
2) the second type requires only a single methylated CpG pair (MeCP2 is an example)
What is involved in histone deacetylation activity?
-sin3 repressor complex
TRUE OR FALSE: methylation is a universal means for controlling gene expression
FALSE: In drosophila there’s very little methylation and in c. elegans in fact there is no methylation. hence there may be some other mechanism besides the methylation State of the DNA that regulates gene expression.
What are three changes that occur in typical active genes?
1) a hypersensitive chromatin site(s) is established near the promoter
2) the chromatin of a domain, including the transcribed region, becomes more sensitive to DNase 1
3) the DNA of the same region is under-methylated
all of these changes are necessary for transcription
How can methylation of CpG islands prevent transcription?
in two ways? methylation of the binding site for some factor may prevent its binding
and
methylation may cause specific repressors to bind to DNA
MeCP1 and MeCP2 are examples of what?
repression caused by proteins that bind to methylated CpG doublets
Fill in the blank: methylation of the promoter ______ transcription
prevents
