Week 7 Flashcards
Why do cells use mRNA as an intermediate rather than directly from DNA?
It helps protect the DNA from damage leading to deadly or inheritable mutations
What is the process of translation/transcription in prokaryotes?
Coupled transcription-translation in the cytoplasm
What is the process of translation/transcription in eukaryotes?
There are different RNA Pols with additional subunits
Transcription produces an pre-mRNA intermediate containing introns that need processing
Translation occurs in the nucleus after which the mRNA is transported to the cytoplasm for translation
Why arent all genes expressed at the same rate at the same place?
Its to do with promoter loading with RNA polymerase II (RNA PolII(2)), chromatin accessibility and interaction with transcription factors
What is a minimum fro RNA PolII to bind to a promoter in eukaryotes?
They require a minimim of seven general TFs in order for RNA PolII to bind to a promoter
What is common for regulatory elements to be located compared to the transcription start site?
It isnt unusual for a gene to have several regulatory elements such as enhancers located many Kb from the TSS?
What are the 3 regions of the classical TATA core minimal promoter required for gene expression?
TATA box
CAAT box
GC box
What are examples of enhancer binding protein that binds to each of the TATA core boxes?
TATA box- TBP
CAAT box- CBP, NF1 and C-EBP
GC box- Sp1
Where is the rough location of each core TATA binding box relative to TSS?
TATA box- ~25 bases upstream
CAAT box- ~80 bases upstream
GC box- ~100 to 200 bases upstream
What is TF II D?
Its a large multiprotein assembly complex that serves as a general TF for transcription initiation by eukaryotic RNA PolII
How does TF II D act as a general TF?
It recognises the core promoter sequences (TATA box) and associated chromatin marks, and interacts with gene-specific activators and repressors
How can proteins be post-translationally modified?
This can be done through the process of phosphorylation and methylation, this can serve many functions including regulation of protein activities
What organisms can post-translational modification of proteins occur?
This occurs in both prokaryotes and eukaryotes but it more extensive so has more regulatory opportunites in eukaryotes
What is the function of RNA polymerase I and III?
RNA polymerases I and III transcribe genes that code for ribosomal RNAs, transfer RNAs, and some small nuclear RNAs.
What is the function of RNA polymerase II?
RNAP II transcribes protein-coding genes and some noncoding RNAs.
How are the genes regulated by RNA polymerase I and III regulated differently than RNA polymerase II?
Promoters recognized by each type of RNA polymerase have different nucleotide sequences and bind different transcription factors.
Genes transcribed by each polymerase have different transcription termination signals and RNA processing mechanisms.
What are chromosome territories?
The area in which a chromosome occupies during interphase staying seperate from other chromosomes
What are interchromatin channels?
Channels between chromosomes that contain little or no DNA and are called interchromatin compartments
Where are transcriptionally active genes are located?
They are located at the edges of chromosomes territories next to the channels of the interchromatin compartments
What is the advatange of placing transcriptionally active genes at the edges of the chromosome territories?
This organization brings actively transcribed genes into closer association with each other and with the transcriptional machinery
What was the advantage of bringing transcribed genes closer to each other and other transcriptional machinery?
This helps facilitate their coordinated expression
What is the function of the interchromatin channels?
They house RNA processing machinery and they are adjacent to the nuclear pore. This helps faciliate capping and splicing and exporting of mRNAs.
How can transcription factories impact nuclear organisation?
Transcription factories impact nuclear organisation by clustering coregulated genes
How are nuclear architecture and transcriptional regulation interdependant?
Changes in nuclear architecture affect transcription, and changes in transcriptional activity necessitate changes in chromosome organization.
How many transcription factories are there?
~100 - 8000 depending on cell type
When and who discovered the first links between open chromatin and active transcription?
It was first 1976 by Harold Weintraub and Mark Groudine
How did Weintraub and Groudine prove the links between chromatin and active transcription?
Weintraub and Groudine isolated nuclei from chicken red blood cells, fibroblasts, and brain cells. They then treated the nuclei with low levels of the enzyme DNase I. This enzyme digests DNA to varying degrees, depending on the chromatin’s conformation. DNA within fully compacted chromatin is shielded or protected from DNase I, while DNA located in regions of more relaxed chromatin is rapidly digested.
What did Weintraub and Groudine do after they used DNase 1 to digest the DNA from red blood cells, fibroblast and brain cells?
They isolated DNA from the nuclei and probed it for the presence of sequences from three genes: betaglobin (transcribed only in red blood cells), avian tumor virus (transcribed in all three cell types), and ovalbumin (not transcribed in any of these cell types).
Betaglobin DNA –> digested in red blood cell nuclei treated with DNase I, but not in treated nuclei of fibroblasts or brain cells.
Avian tumor virus DNA –> digested in nuclei treated with DNase I from all three cell types
Ovalbumin DNA –> not digested in any of the cell nuclei.
How can nucleosomes can be modified?
By changing their histone complex, mostly contain H2A but using varient histones such as H2A.Z influence transcription
How does the introduction of the histone H2A.Z to the nucleosomes impact its function?
Nucleosomes are generally a physical barrier to RNA polymerases and DNA-binding transcriptional regulators, nucleosomes containing the H2A.Z variant are not as stable and thus are less of a barrier.
What is method for chromatin alteration?
Chromatin alteration involves histone modification. Histone modification refers to the covalent addition of functional groups to the N-terminal tails of histone proteins. The most common histone modifications are added acetyl, methyl, or phosphate groups.
How does acetylation allow for binding of transcription regulatory proteins to DNA?
Acetylation decreases the positive charge on histones, resulting in a reduced affinity of the histone for the negative charges on the backbone phosphates of DNA. This in turn may assist the formation of open chromatin conformations.
What catalyses histone acetylation?
Histone acetyltransferase (HAT) enzymes
How are HAT enzymes recruited?
HATs are recruited to genes by the presence of certain transcriptional activator proteins that bind to transcription regulatory regions.
Transcriptional activator proteins themselves have HAT activity.
What are histone deacetylases (HDACs)?
They remove acetyl groups from histone tails
What recruits HDACs?
HDACs can be recruited to genes by some transcriptional repressor proteins that bind to gene regulatory sequences.
What is chromatin remodelling?
The repositioning or removal of nucleosomes on DNA, this is bought about by chromatin remodelling complexes
What are chromatin remodelling complexes?
Chromatin remodeling complexes are large multi-subunit enzymes that use the energy of ATP hydrolysis to move and rearrange nucleosomes.
What is the impact of repositioning of nucleosomes?
Repositioning of nucleosomes makes regions of the chromosome accessible to transcription regulatory proteins, such as transcriptional activators, and RNAP II.
What is an example of remodelling complexes?
SWU/SNF
How do remodelling complexes work?
They can act in several different ways:
Loosen the association between histones and DNA, resulting in the nucleosome sliding along the DNA and exposing regulatory regions.
Loosen the DNA strand from the nucleosome core, or they may cause reorganization of the internal nucleosome components such as swapping in and out histone variants.
Where does DNA methylation most often occur in eukaryotes?
Position 5 of cytosine. Methylation occurs most often in the cystine of CG doublets in DNA, usually on both strands
What happens with the Methyl group and DNA?
The methyl group protrudes into the major groove of the DNA helix.
What is the distrubution of CpG sequences?
They are not randomly distrubuted but tend to be concerntrated in CpG-rich region called CpG islands. Roughly 70% of promoters in human genes have a CpG island in the promoter sequence
What is the first evidence for relationship for methylation in eukaryotic gene expression?
Inverse relationship exists between the degree of methylation and the dregree of expression
Large transcriptionally inactive regions like the X chromosomes in female mammalian cells are heavily methylated
What is the second evidence for relationship for methylation in eukaryotic gene expression?
Methylation patterns are tissue specific and once established are maintained for all cells of that tissue
Proper patterns of DNA methylation are essential for normal mammalian development, which can be altered by methylation and demethylation enzymes in order to silence or activate the transcription of genes in such region
What is the most direct evidence of a role for methylation in gene expression?
Using base analogs such as 5-azacytidine being incorporated into DNA in place of cytidine during DNA replication.
Base analogs can’t be methylated causing undermethylated of the sites where it is incorporated. The introduction of base analogs changes the pattern of gene expression and stimulates the expression of alleles on inactivated X chromosomes.
The prescence of 5-azacytidine in DNA can induce the expression the expression of genes that would normally be silent in certain differentiated cells
What is the % of methalyed cystine in key species?
14%- Arabidopsis thaliana
4%- Mice
1%- Humans
Absent in Caenorhabditis elegans
What are core promoters?
The minimum part of the promoter needed for accurate initiation of transcrption of RNAPII.
~80 base pairs long and include transcription start site
What are proximal promoter elements?
Generally located up to ~250 nucleotides upstream of the transcription start site and contain binding sites for sequence-specific DNA binding proteins that modulate the effciency of trancription
What are the 2 types of core promoters?
Focused core promoters- specify transcription initiation at a single specific start site
Dispersed core promoters- direct initiation from a number of weak transcription start sites located over a 50 to 100 nucleotide region
What is thedifference in promoters between lower eukaryotes and verterbrates?
Most genes of lower eukaryotes use focus transcription intitation where as 70% of verterbrate genes use dispersed promoters
What is the difference in promoters with expression frequency?
Focus promoters are usually associated with genes whose transcriptional levels are highly regulated in terms of time and place
Dispersed promoters are associated with genes that are transcribed constitutively, housekeeping genes, which are needed in almost all cell types
What are the relationship between core promoters and species?
There is no universal core promoter sequence between species and between genes within a species
What is the initator element (Inr)?
It encompasses the transcription start site from approximately nucleotides -2 to +4, relative to the start site
What is the initator element in humans?
The Inr consensus sequence is YYAN(A/T)YY.
Y = any pyrimidine nucleotide
N = Any nucleotide
What is the consensus sequence for TATA box?
TATA(A/T)AAR
R = Any purine nucleotide
Where is the TATA box located relative to transcription start site?
Approximately -30 nucleotides
Where is the TFIIB recognition element (BRE) located?
Either immediately upstream or downstream form the TATA
Where is motif ten element (MTE)?
Located downstream of transcription start site approximately 18 to 27 nucleotides
Where is the downstream promoter element (DPE)?
Downstream of transcription start site by 28 to 33 nucleotides
What is the relationship between the different initiator elements and the promoters?
Versions of BRE, TATA and Inr elements are universal
MTEs and DPEs are found in only found in some of these promoters
What is the function for core promoter elements?
They serve as a platform for the assembly of RNAP II and general transcription factors, which is a critical step in gene expression
What is the function of proximal promoter elements?
They act along with core-promoter elements to increase the levels of basal transcription
What is the CAAT box?
It is a common proximal-promoter element
Where is the CAAT box located?
70 to 80 base pairs upstream from the transcription start site
What happens if there is a mutation in the CAAT sequence?
Mutations within the CAAT sequence dramatically lower the rate of transcription. Thus, genes with a CAAT box, it is required for robust transcription
What is the GC box?
Another element often found in promoter regions and has another element found in promoter regions and has the consensus sequence GGGCGG
What is specific order for the assembeling of the pre-inititation complex?
TFIID binds to the TATA box on the basal promoter region.
The binding of TFIID to the TATA is facilitated by TFIIA. TFIIA facilitates the binding by interacting with the central subunit of TFIID (TBP - TATA box binding protein) to form a complex.
TFIIB is recruited and this interacts with TFIID by also interacting with the TBP subunit of TFIID.
The addition of TFIIB allows the recruitment for RNA Pol II to the complex. RNA Pol II comes together with TFIIF to the complex.
The preinitiation complex is completed with the addition of TFIIE and TFIIH. TFIIE binds to the complex first. The binding creates a site for TFIIH dock onto and completing the formation of the complex
Hydrolysis action occurs as TFIIH helicase activity separates the template strands.
As the promoter melts, TFIIA and TFIID stay behind while TFIIB, TFIIE and TFIIH are released. TFIIF moves down the template along with PolII
What is the CpG island?
A repatable section of DNA containing anywhere from >200bp up to several Kb of Cs and Gs
What is the function of the CpG island promoter?
~50% of mammalian TSS are contained within CpG islands. These have evolved as important regulatory structures in genomes where DNA methylation plays a regulatory role
What is the function of the CpG island promoter?
~50% of mammalian TSS are contained within CpG islands. These have evolved as important regulatory structures in genomes where DNA methylation plays a regulatory role
What happens with the majority of CpG island promoters?
They do not contain TATA boxes
The majority of promoter CpG islands are unmethylated
What was previously thought to be the major-limiting step in transcription?
The recruitment of RNA PolII
What was wrong with thinking RNA PolII was the limiting step?
RNA PolII is already bound to CpG island promoters at many inactive genes
What happens at transcriptional elongation?
The GC box is used as a template which attratcs the TF of Sp1
Sp1 then attracts RNA PolII
The RNA PolII switches from a Serine 5 phosphylation to a Ser 2 phosphorylation
How does TFs work?
They can work alone or as part of a complex to regulate transcription by influencing the recruitment of RNA PolII to a specific gene
What is the defining feature of TFs?
They contain DNA-binding domains which allow for specific attatchment to specific DNA sequences. This allows them to be grouped often on DBD characteristics
What are enhancers?
Are cis-regulatory elements (CREs) comprising short DNA sequences that bind TFs to increase the likelihood that transcription occurs
What is the relationship between the enhancer and the genes it interacts with?
They can be located at a great distance from promoter of a gene and can interact with several genes. They can be either upstream or downstream of their target promoter and do not possess promoter function
What are super enhancers?
They are regions comprising multiple enhancers ensuring high density og TFs and high levels of transcription
What are the similarities between helix-turn-helix and leucine zippers?
They form dimers that ass extra complexity and versatility. The formation of different heterodimers and homodimers means three distinct complexes can be formed from proteins
What is the structure of Zinc-Finger TFs?
They have a secondary structure (alpha-helix and beta-sheet) held togther by the Zn ion. The alpha-helix contains 2-4 tandem domains that recognise specific sequences in the major groove of DNA
How many genes are influenced by MYC protooncogenes?
Influence around ~15% of all genes
What is the function of MYC?
MYC has a low affinity for E-Box regulatory sequences, to be active it must dimersise with MAX.
What can the MYC/MAX complex recuit?
The MYC/MAX heterodimer recruits chromatin-modifying transcription activators including histone acetyl-transferases (HATS)
What does MAD/MAX complex recruit?
The corepressor complex containing histone deacetylases (HDACs)
What determines the balance fir transcriptional activation and repression?
The ratio of MYC to MAD
When is DNA most compact?
During metaphase
What is DNA organised into?
DNA is organised into chromatin which is mediated by histone proteins forming nucleosomes
What is each nucleosome composed of?
Two H2A, H2B, H3 and H4 to form a histone octamer
Two full turns of DNA approximately 146bp of DNA
Whta extends out of the nucleosome core?
A N-terminal tail and can be modified influencing structure
What methods are there for determining chromatin accessibility?
FAIRE
DNase
ATAC
What were the results of using FAIRE, DNase and ATAC?
That 1-2% of the human genome is open chromatin
What is the classic definition of epigenetics?
A change in phentoype with a change in genotype
What is the modern definition of epigenetics?
The study of heritbale chnages in genome function that occurs without a change in DNA sequence
How did Denise Barlow refer to epigenetics as?
The punctuation of the genome, it organises th letters of the DNA code to be functional
What is a key example of epigenetic changes in a cell?
Cell differentiation- all cells have the same DNA but they are expressed differently
What is DNA methylation?
The addition of a methyl group to cytosine bases of CpG dineucleotides
What are enzymes that add methylation to DNA?
DNMT3 A and B de novo
DNMT1 - maintenance during replication
What are enzymes that remove methylation?
TET1-3
What is the method for adding methyl groups to cytosine?
Cytosine is converted to 5-methylcytosine
Using SAM as a methyl donor forming SAH
What is the method for removing methyl group?
5-methylcytosine is converted to 5-hydroxymethylcytosine
Using alphaKG as hydroxy donor forming succinate
Why does adding a hydroxy group to the methyl group remove it?
As it turns into a hydroxymethyl which prevents it from being recognised during replication
What is the relationship between CpG and methylation?
The majority of the 28 million CpG in the mammalian genome are methylated
Why is C to T mutation over represented?
Due to spontaneous deamination of C to T
Where are CpG rich regions commonly associated?
CpG islands are ofte associated with the promoter of genes, where selection pressure does not allow the spontaneous mutation of C to T
What is the relationship between CpG islands and methylation?
The majority of the 40,000 CpG islands are unmethylated
What is the C-terminal domain of DNMT?
It is the catalytic domain
What is the function of DNMT3L?
It is so truncated that it has no catalytic activity but is a co-factor for other DNMTs by forming a complex
What is the function of DNMT1?
Copies the epigenetic changes from the template to the newly constructed strand during DNA replication
What is the function of DNMT3 A and B?
It is de novo meaning it doesnt need a template to add methyl groups
What areas of the genome are highly methylated?
Methylation silences LINE-1, Alu repeats and HERVs to stop the jumping around the genome. The genome becomes unstable if these are unmethylated eg in cancer
Gene bodies (not promoters) are highly methylated to stop suprious transcription initation and the production of potentially deleterious transcripts/truncated proteins
How does DNA methylation influence transcription?
Some Tfs are methylation-sensitive eg CTCF but methylated DNA also has repressive histone modification and closed structure
What is the chromatin structure?
Postively charged histones tightly bind and organise negatively charged DNA to form nucleosomes
How can N-terminal tails of the core histones can be modified?
Methylation, acetylation, phosphorylation and ubiquination
What keeps enhancers open to chromatin allowing for TF binding?
H3K27ac
H3K4me1
What are the two types of chromatin?
Euchromatin- transcriptionally active
Heterochromatin - transcriptionally inactive
What are the two types of heterochromatin?
Constitutive heterochromatin - permenately inactive
Facultative heterochromatin - Flip between eu and heterochromatin
What is euchromatin transcriptionally active?
Acetylation neutralises the positive charge on histones and decreases the interaction with the charged phosphate groups of DNA. The DNA is unmethylated and enriched for the permissive H3K4me2/3
Why is euchromatin lacking heterochromatin epigentic marks?
This helps make the underlying DNA sequence accessible to transcription machinery
What mediates facultaive heterochromatin?
Polycomb-mediates silencing (H3K27me3) is a fine balance between HMT and HMDs and allows for quick induction of expression
When is polycomb-mediated silencing?
During early stages of embryonic development due to decide cell fate
Where does polycomb-mediated silencing occur?
Occurs primarily at unmethylated CpG island promoters. Can be bivalent with active histone marks
What is the order for Higher-order chromatin interactions?
Territories –> compartments –> TADs –> Sun-TADs –> Loops –> Nucleosomes
What is the function of Chromatin Confirmation Capture (3C)?
A techniques that allows for long-range DNA contacts to be mapped
What is the method for Chromatin Confirmation Capture (3C)?
Chromatin crosslinking- Freezes DNA where it is
Digestion- Restriction enymes break DNA apart
Ligation- Rebuiild the loops back together (can be bufferd with PCR)
Purification- Purify the DNA back to remove overhangs
What is the resolution of TADs and Sub-TADs?
TADs are relatively constant in all tissues
Sub-TADs are dynamic, containing the cell-specific enhancer-promoter interactions
Why is Fragile-X syndrome caused by CGG^n repeat?
CGG expansion occurs during female meiosis so a mother with a premutation may have an affected child with a full mutation. CGG repeats beyond 200 units become methylated, silencing the FMR1/FMRP gene
How is Rett Syndrome caused by methylation?
Mutations in the MeCP2 gene
MeCP2 specifically binds to methylated CpGs and coordinates gene repression
