MCB*2050 Midterm Prep
TAR
Transactivating RNA element - part of the Pol II complex - TAT binds to it and prevents elongation
Immuno-precipitation
- technique with antibodies - we can hook the antibodies to large beads and mix them with extract, then wash away the extract. The antigen (and its associated proteins) remain associated with the beads via the antibody - two approaches: Pre-immobilized approach or Free-antibody approach
Immuno-fluorescence
- technique with antibodies - use specific antibodies with a fluorescent dye and localized the antigen in the cell
overview of mRNA processing in Eukaryotes
1.) Transcription, 5' capping 2.) Cleavage at poly(A) site ( done with endonuclease) 3.) Polyadenylation (done with Poly(A) polymerase (PAP) and ATP) 4.) RNA splicing - pre-mRNA processing events are co-transcriptional - end modifcations protects mRNA from degradation - The phosphorylated form of CTD (C-terminal domain) of RNA pol II associates with numerous factors engaged in RNA processing: o Capping enzymes o Processing factors (SR proteins) o Cleavage/ polyadenylation factors
Multiple promoter regulatory elements are not found in...
yeast
Zinc finger
- motif example - results when a zinc ion forms bonds with two cysteines and two histidines - these fingers often come in pairs - the hydrophobic amino acids are the linkers between the paired zinc fingers
Leucine zippers
- motif example - two alpha-helices that are not linked - the leucines interact and bind with each other that end up zipping the helices together
NELF
- negative elongation factor - when bound, the transcription in eukaryotic cells cannot be elongated
Eukaryotic Gene Control
- purpose is the execution of precise developmental programs so that the proper genes are expressed in proper cells at the proper times during embryologic development and cellular differentiation - transcription takes place on DNA that is wrapped up to form chromatin - chromatin needs to be open for transcription to proceed - chromatin-mediated regulation can change gene transcription by regulating promoters - once the gene is in open chromatin, a very elaborate system and a variety of factors regulates the expression of each individual gene
sigma-factors
- recognize promotor elements (-35, -10) and load RNA polymerase at the start site
EMSA (electrophoretic mobility shift assay)
- running electricity through a buffer forces DNA molecules to move towards the positive electrode - the DNA molecules are negatively charged large molecules - if the electrophoresis is performed in a gel, the gel retards the molecules according to their size (the larger the molecule, the slower it moves) - If a DNA fragment binds a protein, the complex becomes LARGER and moves SLOWER
General Polymerase II Transcription Factors (GTFs)
- several of these assemble the preinitiation complex - TFIIA, TFIIB, TFIID, TFIIE, and TFIIH - includes DNA helicase, protein kinases, other factors that help the polymerase elongate and other factors that move nucleosomes out of the way
Gene expression in Eukaryotes
- the activator factors form a scaffold - the activators need to be in the proper conformational state in order for transcription of the desired gene to be initiated - when NELF is bound, transcription cannot be elongated -DSIF can act as a promoter or repressor and is required to prime for transcription - P-TEFb displaces the NELF so that elongation can begin and active gene transcription
Clamp domain of eukaryotic polymerases
- the clamp domain is what clamps onto the DNA and is closed by a bridge - the bridge is part of the clamp domain
lac operon (+ lactose, +glucose)
- the lac repressor is activated in the presence of lactose - the 4 binding sites of the lac repressor are each bound by lactose - this releases the lac repressor from blocking the transcriptional initiation site - transcriptional rate is low because there is still a presence of glucose and therefore, the cAMP isn't bound - glucose is still being metabolized
The mediator complex and RNA pol II
- the mediator complex links activators with RNA pol II - Acts as a bridge between transcriptional activators and RNA Pol II to stimulate RNA synthesis - Brings distal promoter/ enhancer elements and bound factors close to the basal promoter region by looping the interviewing DNA - Mediator is a multi-subunit complex (~30 subunits) - Different subunits interact with specific transcription factor activation domains - One of the subunits has histone acetylase activity to maintain nucleosome-free DNA - Other subunits contact general transcription factors and RNA Pol II to assist in the assembly of the pre-initiation complex
Enhancers in the Regulatory Sequences in Protein-Coding Genes
- these can be found within the transcription sequence and more often than not they are in the intron sequence - they can also be found up stream or down stream from the initiation complex
Promoters in the Regulatory Sequences in Protein-Coding Genes
- they direct the binding of RNA polymerase II to DNA - they determine the site of transcription initiation and influence the frequency of transcription initiation
Multiple domains of the transcriptional activators and repressors
- they have independent DNA-binding domains and effector domains (activation or repression) - these can be identified by deletion analysis of the protein of interest - DNA binding domains have conserved motifs (structure folds)
How do GTFs recognize promoters?
- they recognize the core promoter sequence in eukaryotic DNA - promoters are regulated by the TATA box and initiator transcription starts at a defined point, usually an A codon the on the coding stand - and the three elements: TATA box, Initiator, and BRE and/or DPE
Transcriptional activators
- they regulate the release of the paused RNApol II by activating P-TEFb (CDK9/CycT) kinase, which phosphorylates and inactivates the factors (NELF, DSIF) that cause the pausing
miRNAs
- transcribed by RNA polymerase II - micro-interfering RNAs - translation control
siRNAs
- transcribed by RNA polymerase II - small interfering RNAs - chromatin-mediated repression and translation control
snRNAs
- transcribed by RNA polymerase II - small nuclear RNA - RNA splicing
7S RNAs
- transcribed by RNA polymerase III - signal recognition particle for insertion of polypeptides into the endoplasmic reticulum
snRNA U6
- transcribed by RNA polymerase III - splice proteins
5S rRNAs
- transcribed by RNA polymerase III - the ribosome component - synthesize proteins
tRNAs
- transcribed by RNA polymerase III - transfer RNA - synthesize proteins
Helix-turn-helix
- two helices that have a linker amino acid sequence - the alpha helices turn and insert themselves and bind the major groups that are next to each other
Communication of Enhancers and Promoters
- upstream enhancers communicate with promoters through DNA bending - these are influencing transcription
Euchromatin
- when stained, it is the light portion of the DNA in the nucleus - this is where most eukaryotic genes are located - genetically active - available for transcription
Heterochromatin
- when stained, these are the dark regions of eukaryotic DNA due to it being more densely packed - rich in repetitive DNA mostly located in centromeres and telomeres, mostly puched against the membrane of the nucleus - generally, not accessible to transcriptional machinery - genetically inactive - contains genes that are not essential for the function of the cell
4 ways that histones are post-translationally modified
1) Phosphorylation 2) Methylation 3) Acetylation 4) Ubiquitinylation - the level of the post-translational modifications
Chromatin Immunoprecipitation (ChIP) with anti-RNA pol II antibodies technique
1) living cells or tissues are treated with a membrane-permeating cross-linker such as formaldehyde 2) Sample is sonicated to shear cellular chromatin to short fragments and add antibody to Pol II 3) Immunoprecipitate is added to isolate Pol II cross-linked to DNA 4) Cross linking is reversed, the DNA is isolate, and subjected to massively parallel DNA sequencing
EMSA protocols
1. Create a radioactive DNA probe containing the promoter sequence 2. Mix probe with cell extract (-/+ treated cells) 3. Separate unbound probe DNA from probe/protein complexes on a non-denaturing polyacrylamide gel 4. Expose the dried gel to X-ray film to detect radioactivity - probe bound to protein migrates more slowly than free probe - protein (transcription factor) binding is sequence specific - identity of the transcription factors (super-shift)
Stability of most mRNAs is controlled by...
by the length of the poly(A) tail and the binding of various proteins to 3' UTR (untranslated) sequences
Promoter-proximal elements and enhancers in the Regulatory Sequences in Protein-Coding Genes are...
cell-type specific
Chromatin-mediated regulation is also known as...
epigenetic regulation
Post-transcriptional gene control
* at the mRNA level · Pre-mRNA is capped, polyadenylated, spliced, and associated with RNPs in the nucleus before export to the cytoplasm · Splicing: a large ribonucleoprotein spliceosome complex catalyzes the joining of two exons and removal of intron · A network of interactions between SR proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that specifies correct splice sites
PCR (polymerase chain reaction)
o Amplifying DNA by repetitive cycles of denaturing and renaturing DNA in the presence of thermostable DNA polymerase (most common one is Taq polymerase) o You need two primers that anneal to the ends of the DNA fragment to be amplified o Taq polymerase synthesizes new strands of DNA using the 3' ends of the primers as starter points o Upon completion of this round of synthesis the DNA strands are denatured at high temperatures. The temperature is then lowered and more primers anneal to the new strands o The cycle is repeated
Massive Parallel DNA Sequencing
- All clusters are sequenced in parallel, but not the chemistry of colour emission - You can assemble a whole genome sequence by computer algorithms for alignment - A sample of RNA is fragmented into small pieces (100 bp) and converted to small pieces of DNA - DNA is sequenced "massively" to generate billions of reads - All these reads are aligned to the known sequence of the genome and the number of reads over each position is plotted - No reads on the graph = gene is not being transcribed - You analyze the sequences of the aligned cDNAs is sense and the anti-sense orientation - The graph tells you that at this position we have synthesis of RNA in both sense and anti-sense orientation
Techniques to analyze binding of transcription factors and histones to DNA
- Chromatin immunoprecipitation (ChIP) - gel-mobility shift
CpG Islands
- CpG islands occupy the promoters for about 70% of genes in vertebrates - the spaces between the different promoter sites are often saturated with CpG islands - these genes are transcribed at a low rate due to cytosine being frequently methylated (transcription can be up-regulated by demethylation of cytosine) - CpG rich DNA contains fewer nucleosomes and is hence easier to transcribe
DSIF
- DRB sensitivity elongation factor - can act as a promoter or repressor in the transcription of eukaryotic genes
How would you find proteins that bind to DNA regulatory elements?
- EMSA: Electrophoretic mobility shift assay
The mating type loci in yeast, HML and HMR
- HML and HMR are constantly repressed - Genetic studies identified several proteins that regulate this tight repression - Rap1 binds next to the repressed loci and recruits Sir proteins - Sir (Silent information region) proteins bind over the repressed locus - Sir2 is a HISTONE DEACETYLASE - All histone H3/H4 in the nucleosomes at HML and HMR are DEACETYLATED - Conclusion: histone hypoacetylation is necessary for the gene repression
Transition to elongation
- In the preinitiation complex, the helicase activity of TFIIH (Ssl2) locally "melts" DNA at the initiation site - TFIIH untwists the DNA so Pol II can get in to begin transcribing - Protein kinase activity of TFIIH-kinase phosphorylates the CTD (carboxy terminal domain) of RNA Pol II and releases it from PIC - NELF and DSIF cause the pausing of the polymerase after initiation. Further phosphorylation of CTD by CDK9/CycT releases the polymerase: elongation begins
The 3 polymerases in gene regulation are:
- RNA polymerase I - RNA polymerase II - RNA polymerase III
Repression from histone hypoacetylation: the telomeres
- Rap1 and Sir proteins are also associated with telomeres - Many telomeres cluster at the periphery of the nucleus and are covered with condensed hypoacetylated nucleosomes - Rap1 binds to the telomeres and recruits Sir proteins - Mutational analyses were conducted on histones - - Lysine --> Arginine (positive charge, cannot be acetylated) gene repression at telomeres is maintained - Lysine --> Glutamine (neutral charge, resembles acetylated Lysine) cannot be deacetylated and gene remains active - Rap1 is the repressor Sir3/Sir4 are co-repressors Sir2 is a Histone-Deacetylase
Sex lethal in drosophila
- Sxl controls its own splicing to produce exons 2,4 mRNA - In males in the absence of Sxl the transcript is spliced into exon 2,3,4 mRNA STOP codon in exon 2 of Sxl - No Sxl protein in males
Transformer proteins in drosophila
- Sxl regulates the splicing of the Tra transcript STOP codon in exon 2 of Tra - only females express transformer - premature codon in exon 2
Three elements that direct the positioning of GTFs of polymerase II in eukaryotes
- TATA box: prevalent in highly transcribed genes - initiator: some genes contain an initiator but no TATA (initiator elements are poorly conserved) - BRE (TFIIB Recognition Element) and/or DPE (Downstream promoter element) also influence the activity of the promoter
Assembly of pre-initiation complex
- a cell needs to produce the specific set of activators required for a promoter/enhancer of a particular/enhancer of a particular gene to express that gene
P-TEFb
- a cyclin dependent kinase - it displaces the NELF so that elongation can begin and active gene transcription of eukaryotes
Motifs
- a frequently found sequence in proteins with a similar function - if they are found, they can typically be assumed that they are a DNA binding domain such as a histone, cysteine, Zinc finger, Leu zippers, helix-turn-helix motif or protien,
carboxy terminal domain (CTD) of eukaryotic polymerases
- a special feature involved in multiple regulatory interactions - in yeast, CTD contains 26 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser, in mammals it contains 52 repeats - the Ser residues in CTD are phosphorylated upon transition from initiation to elongation
lac operon (+ lactose, - glucose)
- absence of glucose will lead to higher concentration of cAMP in the cell which will bind upstream to the transcription initiation site at the catabolite activator protein (CAP) - this will enhance transcription (faster rate) - RNA polymerase binds to -10 position relative to the transcription initiation site - the sigma factor is required for the initiation of transcription - the sigma factor associated with -30 position (30 nucleotides upstream of the transcription START site)
PAX6
- an example of complex regulation of eukaryotic genes - it has multiple enhancers as well as alternative promoters - all three transcripts are produced by all the tissues it is expressed in - complex enhancers direct gene expression during development - PAX6 expression in day 10.5 embryo in the retina and the pancreas is regulated by the green enhancer element upstream of exon 0 - PAX6 expression in the day 13.5 embryo in the retina is regulated by the orange enhancer element around exons 5, 6, 7
Enhancer Properties (RNA Pol II)
- can function over a long distance (~ over tens of kbps from transcription initiation site) - can be upstream, downstrean from the start or within introns - position independent: usually still functional when moved - Orientation independent: function in either normal or the inverted orientation
Combinitorial binding
- common in gene families (e.g. involved in the same metabolic families) - Very often DNA-binding proteins work as dimers - Each monomer can recognize a specific DNA element, but cannot work on its own, it will bind to this DNA only in a complex with a similar protein - If you have three monomers that can form dimers, you can have six combinations of these proteins: AA, BB, CC, AB, AC, BC - These six combinations can recognize six different DNA binding sites - Complicate the situation further by adding an inhibitor for one of the three proteins increase the possible combinations
Consensus sequences of DNA response elements are recognized in two ways
- complimentary sequence but anti-parallel: forward sequence on the 5' to 3' strand, the reverse sequence is on the 3' to 5' strand - complimentary sequence is found twice on the 5' to 3' strand: same sequence, repeated
Cooperative Binding
- e.g. NFAT and AP1 dimerize and attach so the binding is stronger and moves faster
Properties of Promoters (RNA Pol II)
- functions within a short distance from the transcription initiation site (~several hundred bps from it) - immediately upstream from the initiation site - position dependent: usually non-functional if moved - orientation dependent: drive transcription in one direction only
lac operon (- lactose, + glucose)
- glucose is the preferred source of energy so when it is present, there are low levels of cyclic AMP (cAMP) and the lac repressor is bound downstream of the promoter site - this inhibits the binding of the polymerase
Insulators upstream of the enhancers
- important for gene function - they are DNA sequence elements that attract a binding protein - they prevent inappropriate interactions between adjacent genes
RNA polymerase I
- in nucleus of eukaryotic cells - transcribes Pre-rRNA (28S, 18S, 5.8S rRNAs) - make ribosome components and synthesize proteins
RNA polymerase III
- in nucleus of eukaryotic cells - transcribes tRNAs, 55 rRNA, snRNA U6, 7S RNA, and other small stable RNAs - they encode for proteins, form a ribosome component, perform RNA splicing, create a signal recognition particle for insertion of polypeptides into the endoplasmic reticulum, and other functions
RNA polymerase II
- in nucleus of eukaryotic cells - transcribes mRNA, snRNAs, siRNAs, and miRNAs - encodes protein, RNA splicing, chromatin-mediated repression, and translation cotrol
Protein Dimerization
- increases the complexity of DNA-binding specificity - 3 different transcription factors from the same family with different binding specificities can be formed from two bHLH (basic helix-loop-helix) monomers - A1-A1 homodimer binds to two A1 DNA elements - A2-A2 homodimer binds to two "A2" DNA elements - A1-A2 heterodimer binds to a combination of A1 and A2 DNA elements - generally created with helix-loop proteins
lac operon in bacteria
- lac operon is a regulated promoter - it is regulated by catabolite activator proteins (CAP) that bind next to a promoter to recruit RNA polymerase - it is repressed by the lac repressor which binds downstream of the transcription initiation site
Shared structure of hormone nuclear receptors
- ligand binding domain: 225-285 aa, and 15-57% conformational similarity - DNA binding domain: 68 aa, 42-94% conformational similarity - variable region: 100-500 aa, 0% similar
mRNA
- messenger RNA - transcribed by RNA polymerase II - encodes protein
Transcription activators and repressors in the Regulatory Sequences in Protein-Coding Genes
- modular proteins containing a single DNA-binding domain and one or a few activation or repression domains
Antibodies as a technique
o Antibodies are natural immunoglobulins produced by animals to combat invading exogenous proteins of any kind (recognition of "self" versus "non-self" proteins) o Upon invasion by a foreign protein (we call it an antigen) a specialized class of B-lymphocytes rearrange the genes at their immunoglobulin loci and produce UNIQUE AND EXTREMELY SPECIFIC ANTIBODIES against the antigen o We can "trick" the immune system of lab animals by injecting them with antigen that we are interested in (transcription factors, histones, modified histones, any other protein) o The animals produce large amounts of antibodies for us o We purify these antibodies from their sera and use them as HIGHLY SPECIFIC REAGENTS FOR THE RECOGNITION OF PROTEINS OF INTEREST o two techniques: immuno-fluorescence and immuno-precipitation
Contemporary techniques of DNA and RNA Sequencing
o Contemporary techniques rely on dNTPs which emit light upon incorporation into DNA o Contemporary techniques can be scaled up to sequence a whole human genome of 3 x 1010 bases (or equally large samples) in hours
TAT
transactivation of transcription - binds to TAR and to part of the Pol II complex - used to prevent the further phosphorylation of CTD and therefore prevents elongation until it receives the proper signal - when it leaves, the transcription event can begin
Regulation of Pre-mRNA processing
· Alternative promoters, alternative splicing and alternative cleavage at different poly(A) sites yield different mRNAs from the same gene in different cell types or at different developmental stages · RNA-binding proteins that bind to specific sequences near splice sites regulate alternative splicing · An example: Sex-lethal (Sxl), Transformer (Tra) and Double sex (Dsx) regulate sexual differentiation in Drosophila o Sxl (lethal in females, not in males) is an RNA binding protein, acts as a suppressor of splicing o Tra is an RNA binding protein; acts as an activator of splicing o Dsx is a transcription activator/repressor
The mediator: a co-activator
· Bridge between transcriptional activators, GTFa, chromatin remodelers · The mediator is a huge complex containing variety of proteins including enzymes and proteins that recognize activators, GTFs and other coactivators · Different mediator complexes contain proteins required for specific genes · The mediator(s) directly interacts with transcriptional activators · One mediator complex can interact with multiple transcriptional activators · The Mediator (s) directly interacts with GTFs
Repression/Activation at specific genes
· Transcriptional repressors often directly interact with a HISTONE DEACTYLASE o Ume6 is a transcription repressor o Sin3/Rpd3 complex is a co-repressor part of the Rpd3L o Rpd3 is a HISTONE DEACTYLASE · Transcriptional activators often directly interact with a HISTONE ACETYL-TRANSFERASE o Gcn4 is a transcription activator o SAGA complex is a co-activators o Gcn5 is a HISTONE ACETYL-TRANSFERASE part of the SAGA complex o Acetylated Histones can recruit more co-activators via bromo-domains
Histone Code
· Various combinations of histone modifications · These modifications recruit factors to "decorate" chromatin in various ways (with different promoters, repressors for example) · It is estimated that there could be thousands of different histone codes - how it is fully duplicated in most cases is unknown
Cytoplasmic control of mRNA stability
· multiple PABPI (poly(A)-binding protein I) molecules bind to the poly(A) tail and interact with initiation factors that bind to the 7-methylguanosine cap · the longer the poly(A) tail, the more PABPI will associate with it · the circular structure of mRNA increases translation efficiency by aiding in the rebinding of ribosome subunits to the translational start site · mRNA stability is controlled by the length of the poly(A) tail o short-lived (c-Fos) contain multiplE copies of the sequence AUUUA in the 3' UTR that interacts with a deadenylating enzyme o long lived mRNAs (globin) contain repeats of the stabilizing sequence CCUCC in their 3' UTR
Lipid-Hormone Receptors/Transcription Activators
· steroid hormones: they can travel through membranes · Lipid-soluble hormones diffuse into the cell through the cell membrane and bind to a soluble receptor · Some receptors are located in the nucleus, the binding of the hormone changes their activity · Some receptors are located in the cytoplasm · Upon binding of the hormone the receptors-hormone complex moves to the nucleus. It is now a transcription factor - it binds to specific response elements in genes to regulate gene expression
Double sex in drosophila
· the transformer influences the alternate splicing and cleavage of Dsx into male and female isoforms · Dsx is a transcription factor binding to DNA with the same DNA binding domain in both male and female isoforms · The alternatively spliced female isoform of Dsx contains a transcriptional activation domain · The alternatively spliced male isoform of Dsx contains a transcriptional repression domain · Slx acts as an intron splicing silencer (inhibitor) · Tra acts as an intron splicing activator
transcriptional activators
· they de-condence chromatin · Transcriptional activators bind and recruit Histone Acetyl-Transferases · Transcriptional activators also bind and recruit another class of co-activators: Chromatin-remodeling complexes · All chromatin remodeling complexes are homologous to the yeast SWI/SNF complex · Once histones are acetylates, nucleosomes become loose with spaces in-between them · Chromatin-remodeling complexes then push them along DNA to "open" promoters
DNA and RNA sequencing
- Uses probes/primers to identify genes of interest - contemporary techniques rely on dNTPs which emit light upon incorporation into DNA
DNA Methylation
1. Methylation of CpG islands is associated with condensation of chromatin o Methylated DNA recruits a special class of Me-DNA-binding proteins (MeBPs) o MeBPs recruit factors that condense chromatin and deacetylate histones 2. Unmethylated CpG islands recruit proteins that methylate a specific position on histones: Histone 3-Lysine 4 (H3-K4) 3. H3-K4 is recognized by the transcription initiation machinery o DNA methylation marks are reconstituted on the new DNA strands immediately after the passage of the replication fork o DNMT (DNA Methyl-Transferase) enzymes associate with the fork and travel with it
Applications of EMSA
1. You can identify promoter sequences required for transcription by deletion mapping. 2. You can measure DNA-binding activity by gel-mobility shift assay. 3. Demonstrate DNA-binding in vivo by chromatin immunoprecipitation ChIP - the gel shift assay tells you whether or not cells contain a transcription factor that is capable of binding to a specific DNA sequence in vitro
Epigenetic regulation of transcription
· Cell differentiate and select the genes they express · Then the cell divides, but express the same genes after cell division · DNA replication is a major disruptive force for chromatin structure and for bound transcription factors. How do the cells "remember" which genes to express? · This continuity of gene expression is achieved mainly through reconstruction of the same type of chromatin after the passage of the replication fork · We call this epigenetic memory of transcription · Epigenetic control of transcription is maintained by DNA methylation and methylation and acetylation of histones · We call these epigenetic marks · Epigenetic marks are faithfully rebuild during and/or soon after the passage of the replication fork · When the replication fork passes condensed chromatin, immediately after that, the cell rebuilds condensed chromatin
Transmission of epigenetic marks
· Cell express the same genes after DNA replication (a major disruptive force for chromatin structure and for bound transcription factors) and cell division · This continuity is achieved mainly through reconstitution of the same type of chromatin after the passage of the fork - we call this epigenetic control of transcription · Epigenetic control of transcription is maintained by DNA methylation or histone methylation
Nucleosome repositioning factor (SWI, SNF)
· Example: SWI/SNF (switching/sucrose non-fermenting) ATPases · Move nucleosomes along the DNA by sliding or transferring them · Shifting nucleosomes away from the promoter/enhancer sites gives transcription factors access to the DNA
Regulatory Sequences in Protein-Coding Genes
· Expression of eukaryotic protein-coding genes is regulated though multiple protein-binding transcription-control regions located at various distances from the transcription start site - promoters, promoter-proximal elements and enhancers, and transcriptional activators and repressors are all factors in this process
The 3 possible marks on the H3 tail
· H3-K4 methylation - associated with active transcription · H3-K9 methylation - associated with condensed chromatin and NO TRANSCRIPTION · H3-K27 methylation - associated with condensed chromatin and NO TRANSCRIPTION
HIV, AIDS and Transcriptional Elongation
· HIV (Human Immuno-deficiency Virus) has a mechanism to suppress CDK9/CycT activity by virally encoded protein called TAT. This pauses RNA polymerase II at its LTR promoter (a weak promotor) · Upon stress, TAT releases CDK9/CycT (which phosphorylates Pol II) and the polymerase transcribes the virus · This is one mechanism of viral latency: it is dormant and released from time to time, slowly killing T-cells and abolishing immunity · The patients then die from any opportunistic infection
Transmission factors: Polycomb and Trithorax complexes
· HOX genes are initially repressed by sequence-specific repressors during embryogenesis · Polycomb Repression Complex 2 (PRC2) maintains repression by continuously methylating H3-K27 through its enzymatic or catalytic subunit (acts first) · Polycomb repression complex 1 (PRC1) recognizes H3-K27-Me and compacts chromatin (acts second) · Trithorax complexes oppose repression by Polycomb complexes by methylating H3 at lysine 4 and maintaining this activating modification during chromosome replication
Histone "tails" and their modifications
· Histone tails "grab" DNA usng the positive charge of the L and R residues · K (lysine) acetylation reduces the positive charge, reduce the grip and facilitates transcription · Histone tail modifications also determine the "histone code" of epigenetics
What determines the height of the peaks in massive parallel DNA sequencing?
· Massive parallel RNA sequencing is showing the intensity of transcription over the analyzed DNA · A powerful computer aligns the sequences of the small cDNA fragments over the known genomic locus · The number of times each sequence is aligned over the sequence determines the height of the peaks
Transcription, DNA repair and Cancer
· Mutations in subunits of TFIIH have been identified as the cause of a specific skin cancer: Xeroderma pigmenotsum · The mutations inhibit DNA repair caused by such things as exposure to UV light · Patients develop multiple skin lesions that develop into cancer · Hence: TFIIH is involved in transcription-coupled DNA repair
Control of gene expression in bacteria
· Prokaryote gene expression is regulated primarily by mechanisms that control transcription · In prokaryotes one enzyme, RNA polymerase, transcribes all genes · A group of proteins called sigma-factors recognize promotor elements (-10, -35) and load RNA polymerase at the start site · An operon is a group of genes that are all transcribed together with one common promoter, so either all of the genes are transcribed or none of the genes are transcribed (e.g. the lac operon) · It is regulated by activator proteins (CAP, Catabolite Activator Protein) that bind next to a promoter and recruit RNA polymerase · Bacterial gene expression is regulated by transcriptional activators and repressors
Assembly of pre-initiation complex on TATA-containing promoters
· Promoter elements (like TATA or the initiator) direct general Transcription Factors to bind DNA · General Transcription Factors position RNA Polymerase II at start sites assist in initiation · Assembly of pre-initiation complex on TATA-containing promoters: · PIC = preinitiation complex is the first thing to assemble (TAFs and a TBP which makes TFIID the PIC) · TFIID binds to the TATA box to initiate transcription · During initiation the CTD (Carboxy-Terminal Domain) of RNA pol II establishes contact with TFIIF and TFIIH and TFIIE · The core PIC = TFIID, TFIIB, TFIIA, TFIIF and Pol II · Can't start initiation until TFIIH attaches to the core PIC · Closed PIC = no helicase activity · Open PIC = The helicase will start unwinding the DNA and get rid of all nucleosomes · The initiation factors and general transcription factors will be exchanged for elongation factors to build the elongation complex
transcription factors activity
· Transcription factor activities are indirectly regulated by cell-surface receptors and intracellular signal transduction pathways: o A cell sends a signal to other cells by expressing and secreting a peptide hormone o This peptide hormone reaches a membrane bound receptor on the surface of the recipient cell o The binding of the hormone initiates a signal transduction pathway (includes Protein Kinases that phosphorylate various proteins) through modifications of several proteins o Eventually, a transcription factor is activatd by phosphorylation. It directs the expression of its target gene
