Dr Robert O.J. Weinzierl Flashcards
What are the main things focused on in Roberts Lecture series?
- Transcriptional Machinery - focus on Eukaryotic
- DNA packing + modification
- Case study - cMyc
General structure of Bacteria RNA polymerase?
How many Eukaryotic RNAPs are there? What functions do they perform?
Subcellular distribution of Eukaryotic RNAPs?
Main things to note…
- RNAP1 is found in the Nucleoli (sub-compartment in nucleus)
- RNAP2 and 3 found in the nucleoplasm
- mtRNAP –> Present outside the nucleus in the mitochondria
Structurally speaking, how does the Bacterial Core RNAPs compare to Eukaryotic RNAPIIs?
As is the case with bacterial RNAPs, the surface of RNAP II is almost entirely negatively charged except for its main channel and the region about the active site, which are positively charged –> drives negatively charged DNA into the active site
What is one of the main differences in between Bacterial RNAP and Eukaryotic RNAPs?
Hint - Specificity
Apart from basal transcription factor require by RNAPII, what other players are important in the regulation of transcription?
Difference between Eukaryotic and prokaryotic promoters?
Prokaryotic promoters are located a lot closer to the gene transcript itself
Whereas…
Eukaryotic promoters can be located proximally or distally
Note - enhancers, even when located far away, still need to interact with the basal initiation complex –> DNA is flexible and allows this to happen
What is one of the main promoter recognition sequences in Eukaryotes?
What is TFIID?
Shape of TATA binding protein (TBP)? How does it interact with DNA?
What’s the role of TFIIA and TFIIB? How can we optimize binding of the complex?
What experimental technique can be used to study basal factor binding to DNA?
Apart from TATA boxes, are there other promoter elements?
Outline the Textbook illustration of the Assembly of RNAPII basal machinery - image attached below.
Role of TFIIF?
TFIIF binds to RNAPII in solution and facilitates delivery of the polymerase to the TFIID-TFIIB-DNA complex on the promoter
Role of TFIIE and TFIIH (last addition before transcription starts)?
TFIIE is important as it allows recruitment of TFIIH
TFIIH has a CDK7 kinase domain for phosphorylation
Does elongation also have additional factors? If yes, what would they do?
Yes - Elongation Factors (EF) –> many of them are chromatin-remodelling proteins - needed to create free DNA for RNA poly complex to transcribe
Can RNA polymerase II be phosphorylated? If yes, where and why is it important?
Apart from phosphorylating the C-terminal domain, what other role does TFIIH play?
How does transcription intiation differ between Bacteria nd Eukaryotes?
Can the basal machinary of RNAPII yield high gene expression?
Apart from promoter regions, what other regions in Eukaryotic DNA are important for transcription?
General information about gene-specific transcription factors?
What are the two main things that a gene-specific transcription factor needs?
What experiment can we run to find out where gene specific TFs bind?
EMSA assay (electrophoretic mobility shift assay) tells us whether a protein binds to DNA BUT does not tell us where –> mixing DNA + DNA binding proteins –> run through gel –> binds = large complex –> move slower
What chemical interactions are involved with Proteins binding to DNA?
Structure of DNA-B form?
If TF does unwind the DNA, how are they able to read/detect the DNA sequence?
What are the standard DNA binding domains
Outline the general strucutre of the Helix-Turn-Helix motif - what are the C- and N-terminal helices responsible for?
Do H-T-H motifs normally form dimers?
What is the Leucine-Zipper domain? Example?
What is the structure of the Helix-Loop-Helix domain? Example of protein that uses it?
Outline the structure of a Zinc-Finger domain.
How many bases do Zinc-finger recognize?
What is the p53 transcription factor? Why is it important?
What are Activation Domains in gene specific transcription factors?
Functions of the activation domains?
Generally speaking, what structure do Activation domains form? Are ther any specific A.A. present?
Apart from the basal transcription machinary and gene specific TFs, what else influences whether a gene gets transcribed?
Why is DNA compaction needed?
- DNA has to be organized so that it is condensed enough to fit in the nucleus
- Providing another way to regulate transcription levels
How is DNA packaged around nucleosomes (histone complex)?
What would we get if we were to run a partial and complete digestion of Nucleosome packaged DNA?
What would we get if were to run a Nucleosome on a SDS-Page gel?
Remember - SDS-page denatures + reduction of Disulphide bridges - leaving polypeptides behind
How are the histones assembled into a nucleosome?
What is the Evolutionary Origin of histones? Do all domains of life have them?
Are nucleosomes structural conserved between Archaea and Eukaryotes?
Yup, There is structural conservation between Archaea and Eukaryotes
- Both have the histone fold/same organisation –> Structurally very similar
What interactions exist between the Nucleosome & DNA?
The extensive interactions between the nucleosomal surface and DNA is required to bend the 146 bp long DNA into two tight circles (this is normally energetically highly unfavorable because of the rigidity of double-stranded DNA!)
Do Nucleosomes show DNA sequence specificity?
Does DNA become distorted when packaged around nuclesomes?
Yes, Major and minor grooves become distorted –> major and minor groove become wider than normal
One of the main reasons why the transcription factors cannot interact with package DNA - no spatial complementarity
What is the role of linker histones?
Linker histones
Linker histones, such as H1 and H5 stabilize interaction between nucleosomes in compacted chromatin - Binds to Linker DNA in order to stabilize it
Linker histones organize the entry and exit points of DNA on the nucleosome
The core nucleosome packages 146 bp, the presence of H1 extends nuclease protection to 168 - 200 bp –> extra protection
After nucleosomal packaging (Beads on a string), what is the next level of DNA packing?
How does the ‘beads on a string’ structure comapre to the chromatin fibre in terms of transcriptional availability?
Does the In-vivo evidence support the idea of the existance of a solenoid structure (chromatin fibre)?
What is important to remember about the Histone protein N-termini?
What amino acid in the Histone N-termini tends to be modified (Acetylation/Methylation)?
What are the enzymes responsible for Lysine acetylation?
What are the effects of acetylation on the chromatin structure?
Other factors that open up/close chromatin structure?
Example of a gene-specific TF that influences chromatin acetylation?
Example of a gene-specific TF that influences chromatin acetylation (repressor)?
What effect does DNA methylation have on chromatin compaction?
Methylation is another important post-translational control mechanism to distinguish between transcriptionally active and passive forms of chromatin
Methylation is more complicated –> sometimes causes activation whereas other times not - acts more like a signal to other proteins
What do the terms Heterochromatin and Euchromatin mean?
Can all the N- and C-termini of all histones (H2A, H2B, H3 and H4) be modified?
All the N and C termini can be modified of all histones
H3 and H4 are more general modifications
However, H2A and H2B tend to be more specialized modifications
Are the Histone tails highly conserved?
Histones tails are highly conserved from Yeast to Humans
The conservation of these tail regions shows their high importance
Are there High Order’s Nuclear Organization above chromatin?
What is c-Myc?
c-Myc is a proto-oncoprotein, encoded by the c-Myc oncogene
60 - 70% of cancers depend on c-Myc! Shows us that cancer cells require specific transcription factor to be mutated.
Oncogene - is a gene with a normal cellular function that has the potential to cause cancer
Proto-oncoprotein is a normal protein with a normal physiological function whereas, we call it a oncoprotein once it has been mutated
Oncoprotein can be created by mutating the protein, or by mutations increasing the expression of the gene
Why is c-Myc associated with cancer?
c-Myc (up-)regulates the expression of a large number of genes involved in cell proliferation by increasing the metabolic rate. Hence, mutations resulting in cancer formation
Examples of metabolic pathways that increase
- Heightened nutrients uptake
- Enhanced glycolysis/glutaminolysis - energy creating pathways
- Elevated fatty acid and nucleotide synthesis
Drugs that interfere with c-Myc are therefore predicted to have a strong therapeutic effect
Consequences of elevated c-myc levels in cancer cells?
Example in Burkitt’s Lymphoma - Why is there an overexpression of c-Myc?
Apart from overexpression, what other change to occurs to c-Myc in cancer cells?
Structure of c-Myc Heterodimer?
What are two potential drug targets to stop the action of c-Myc heterodimer?
Apart from the DNA binding domains in c-Myc, are we able to target another other part of the protein?
What are Intrisically disordered proteins? What are its common characterisitcs?
Intrinsically disordered proteins Functions?
Is there a degree of conservation in the c-Myc protein across species? Why would this be important to consider?
Have we had any sucess with designing Therapeutic Targets for c-Myc intrinsically disordered region?
Could we predicting c-Myc structure by computational Simulation?
YEAHHHH BUDDDYYY
What promoter region do housekeeping genes (genes expressed in all tissues) tend to have?
Housekeeping genes - have one or more copies of the sequence GGGCGG or its complement (the GC box) located upstream from their transcription start sites
Do regions between -50 to -110 alsp have promoter elements? If so, give an example.
The gene region extending between about –50 and –110 also contains promoter elements
Many eukaryotic structural genes, including those encoding the various globins, have a conserved sequence of consensus CCAAT (the CCAAT box) located between about –70 and –90 whose alteration greatly reduces the gene’s transcription rate.
Futhermore…
Globin genes have, in addition, a conserved CACCC box upstream from the CCAAT box - associated with transcription
Difference between enhancer and promoter element?
Promoter element - associated with ther gene in order to trigger site-specific and strand-specific transcription initiation
Enhancer element - can be located far away (position does not have to fixed) and can have a different orientation (strand) relative to transcribed region
Is the mechanism by which RNAP2 recognizes promoter DNA and intiates transcription conserved across all eukaryotic organisms?
Preintiation complex (PIC) - Including Pol and the whole TFII gang have been found across a wide range of erukayotic species - yeast, fly, rat, human and other metazoan cells
Suggesting that the general transcription factors are universal Pol II transcription factors, and that the mechanism by which Pol II recognizes promoter DNA and initiates transcription is conserved among eukaryotic organisms.
How in the PIC nucleate if there is no TATA box for TFIID to bind to?
Although most yeast promoters contain a TATA box…
Many (perhaps most) metazoan promoters do not, and for these, PIC assembly is nucleated (created) by one of the other promoter elements binding to TAF subunits.
For example, TAF1 and TAF2 bind Inr elements, whereas TAF6 and TAF9 bind DPE elements.
Accordingly, the structural complexity of TFIID is a consequence, in part, of its requirement to recognize a variety of distinct core promoter elements as the first step in PIC assembly
Structural information about TFIIH?
TFIIH is a….
- 10 subunit complex
- With a total mass of 500 kDa
- With several enzymatic activities
a) Two ATP-dependent DNA helicases with 3 ́ → 5 ́ or 5 ́ → 3 ́ polarity
b) Pol II carboxy-terminal domain (CTD) kinase–cyclin pair
What are the two main functions performed by TFIIH?
TFIIH performs critical roles at initiation and at the initiation– elongation transition.
- At initiation, TFIIH facilitates promoter melting - Two Models…
a) One model, XPB (helicase - subuinit) contacts DNA both upstream and downstream of the start site, with ATP hydrolysis inducing a conformational change in XPB that separates the two strands of DNA.
b) The second model proposes that XPB contacts DNA only downstream of the start site and catalyses ATP-dependent rotation of downstream DNA relative
to a fixed upstream site, in essence functioning as a ‘wrench’ to unwind DNA.
- TFIIH regulates the transition from initiation to elongation by Cdk7 catalysed phosphorylation of residue Ser5 of the Pol II CTD
What is the Mediator Protein - Transcriptional intiation?
General transcription factors + Pol II is suffiecient for accurate transcription initiation but this complex fails to interact with activator proteins bound to enhancer or upstream activation sequences.
Mediator is an enormous complex - at least 24 subunits with a total mass greater than 1 MDa –> in yeast revealed that Mediator is required for transcription from most Pol II promoters
Might seem like it is a general TF - but it is dispensable for promoter recognition + Mediator protein does show some level of specificity
Function
- Mediator appears to function as a ‘control panel’ that integrates regulatory signals, often from several enhancer-bound activator proteins, and transduces this information to Pol II and the general transcription factors.
- Mediator associates with the hypophosphorylated IIA form of Pol II and stimulates the CTD kinase activity of TFIIH.
but. .. - A larger form of Mediator includes a fourth complex (Med12, Med13, Srb10, Srb11) with cyclin-dependent CTD kinase activity that inactivates Pol II and TFIIH.
What happens during the transition between the intiation to elongation complex in trnascription?
After the synthesis of a certain length of RNA, the GTFs are replaced with elongation factors (EFs). Thus, the IC isomerizes into a processive elongation complex (EC),
The elongation complex serves as a platform for regulation and various transcription-coupled events, such as mRNA processing, chromatin remodelling, and DNA repair
What are the conserved basal elongation Factors associated with RNAP2? What are their functions?
Conserved basal EFs - Identified using x-ray crystallography and cryo-EM
- Spt4/5 –> a heterodimeric complex of Spt4 and Spt5, is implicated in processive transcription elongation, promoter-proximal pausing in higher eukaryotes, many transcription-coupled events
Note - KOW in image are Spt5 domains
- Elf1 (ELOF1 in human) –> is a small protein conserved in eukaryotes which accompanies transcribing Pol II in vivo
Function:
As Elf1 slightly delays transcription elongation in K. pastoris Pol II in vitro - it seems to directly interact with Pol II and function in transcription regulation
- TFIIS –> induces mRNA cleavage by enhancing the intrinsic nuclease activity of RNA polymerase (Pol) II - TFIIS-induced RNA cleavage can promote proofreading by Pol II
How?
- Acidic residues in the TFIIS loop complement the Pol II active site and position a metal ion and a water molecule for hydrolytic RNA cleavage
- TFIIS also induces extensive structural changes in Pol II that would realign nucleic acids in the active center - facilitate cleavage reaction
What are examples of covalent histone modifications?
Covalent HPTMs such as…
- Acetylation
- Methylation
- Phosphorylation
- Ubiquitination
- SUMOylation
- ADP-ribosylation
- Deimination
- Non-covalent proline isomerization
Can all affect the condensation of chromatin as to organize the genome into transcriptionally active and inactive regions
Are all histone N-terminal tails and globular domains are subject to modification?
Yes, All histone N-terminal tails and globular domains are subject to modification.
Most researched are…
Smaller covalent modification’s methylation, acetylation, and phosphorylation
What are the different types of methylation possible on the histone protein?
Hint - Lysine & Arginine residues
- Lysine residues can be mono-, di-, and trimethylated
- Arginine residues can only be mono- or symmetrically or asymmetrically dimethylated
Common Acetylation & methylation stats associated with heterochromatin and Euchromatin?
Euchromatin - Characterized by…
- High levels of acetylation
- High levels of H3K4me1/2/3, H3K36me3 and H3K79me1/2/3.
Heterochromatin - Characterized by…
- Low levels of acetylation
- High levels of H3K9me2/3, H3K27me2/3 and H4K20me3
What does ChIP-seq data from the Zhao lab reveal about methylation stats and acetylation stats of actively transcribed genes?
Actively transcribed genes are characterized by high levels of…
- H3K4me3, H3K27ac, H2BK5ac and H4K20me1 in the promoter
- H3K79me1 and H4K20me1 along the gene body
Until recently why was it hard to understand the way HPTMs interact with one another to control transcriptional activity? What was used to solve this?
Until recently, elucidating the mechanisms by which HPTMs interact with one another to control transcriptional activity has been complicated due to the layered complexity of combinatorial crosstalk (Modification interacting with each other influencing the net result)
ChIP-seq data and associated gene expression profiles has speedily facilitated decipherment of the histone code (net - histone modification) and its effect on transcriptional activity
What are three broad effects on transcription that can be attributed to Histone post translation modifications (HPTM)?
What are the 5 families of Type-A HATs?
Do HATs work in isolation or are they normally part of a complex?
How many classes of HDACs are there?
General information about histone methylation?
Generally speaking, how do the following methylation events influence transcription?
H3K4 Methylation
H3K36 Methylation
H3K79 methylation
H3K27 methylation
What is histone crosstalk refer to?
Histone modifications can affect multiple cellular processes, which subsequently affects the capacity for the creation or removal of other HPTMs
Basically, the histone modification do not occur in isolation rather they all influence eachother - intricate choreography
What factors lead to an overexpression c-Myc in cancer cells?
The current dogma - MYC amplification as the primary driver in disease states –> e.g. translocation from chromosome 8 to 14 resulting in the gene coming under control of the very active immunoglobulin H promoter
But… Post-translational regulation of MYC has emerged as an important mechanism, irrespective of increased expression
Elevated levels of pS62-MYC (serine phosphorylation) and lower levels of pT58-MYC (Threonine phosphorylation), consistent with a more active and stable form of MYC was found large percentage of tested human tumours!
Mutation of the Threonine 58 (T58) residue - increased tumorigenic potential –> as phosphorylation at Threonine 58 is associated with MyC turnover (degradation)
Oncogenes such as HER2 and mutant KRAS that can enhance S62 phosphorylation
Is downregulation of c-Myc associated with toxicity?
What drug research is available on transcription inhibition of c-Myc?
Hint - BET inhibitor
c-Myc elusive structure - epigenetic silencing seems promising
Extra-terminal motif bromodomains (BET) - BET inhibitors being the most well-studied.
BET family member BRD4 recruits positive transcription elongation factor b (P-TEFb) to promoters and enhancers, promoting the release of RNAPII and initiating transcriptional elongation
Drug?
JQ1, a BET inhibitor, has been shown to inhibit BRD4 binding at MYC promoter and enhancers - decreasing MYC expression in-vitro
Work Clinically? In-vivo?
Results less consistent
Future?
A new BET inhibitor now in clinical trials, BI 894999, reduces tumor growth in vivo and synergistically induces cell death when combined with CDK9 inhibitors
What drug research is available on transcription inhibition of c-Myc?
Hint - G-quadruplexes
Normally DNA G-quadruplex structures resides upstream of the transcriptional start site and silences gene expression
Potential target - Stabilize these structures upsteam of Myc gene?
Results?
Small molecules (GQC-05, Cz1, IZCZ-3, and DC-34) are capable of binding and stabilizing G-quadruplexes within the NHE III region of the MYC promoter, resulting in the suppression of MYC messenger RNA (mRNA) and protein
Problems?
- Clear that these molecules exhibit a MYC-dependent phenotypes but the off-target effects of these various compounds are still being interrogated
- Formation of i-motifs, which form on the opposite strand of G-quadruplexes, can promote MYC transcription - more information needed about the dynamic relationship between these two DNA structures
What drug research is available on heterodimer inhibition of c-Myc?
Targetting bHLHZ domain in C-Myc –> allows for dimerization of MYC to its binding partner MAX and subsequent DNA binding at E-box sequences
Results?
- Inhibitor MYCMI-6 was shown to bind directly within the bHLHZ domain and disrupt MYC/MAX dimerization at low concentrations –> Promising In-vivo and in-vitro results
Downside –> Does not lead to MYC degradation so any actions that are independent of MAX –> may still have an effect
- Struntz et al. demonstrated that MAX:MAX homodimerization stabilization using small molecule (KI-MS2-008) leads to MYC degradation & reduced expression
Reduces MYC levels using an inert MAX:MAX homodimer
Problem?
Binding of other MYC network proteins to E-box sites inhibited - toxicity?
What drug research is available on inhibition of c-Myc PTM?
We know that Serine 62 phosphorylation - stabilizes C-Myc structure
- There are several Kinases inhibitors that could potentially be used –> ERK, CDK2, and CDK9 inhibitors
Unfortunately…
Cancer cells are quite adept at rewiring signaling pathways in response to targeted therapies in order to keep MYC active
- Decrease pS62-MYC is through the activation of PP2A-B –> serine/threonine phosphatase that targets pS62
Studies have shown that B subunit (B56α) has the ability to dephosphorylate pS62 c-MYC leading to decrease levels of c-MYC
Activation of PP2A has emerged as an attractive therapeutic
