week 4+5 - Regulation of Gene Expression in Eukaryotes

Question 1

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
gene expression in eukaryotes

Answer 1

chromatin
-transcription-> primary RNA transcript
-RNA processing-> mRNA
-RNA export-> mRNA in cytosol
-translation-> translated protein
-post-translation processing-> functional protein

Question 2

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
protein degradation

Answer 2

protein degradation plays important role in determining steady state of proteins

Question 2

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNA polymerases:
RNAP I

Answer 2

o Synthesises rRNA (not 5s RNA)

Question 3

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNA polymerases:
RNAP II

Answer 3

o Synthesises mRNA

Question 4

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNA polymerases:
RNAP III

Answer 4

o Synthesises small cellular RNAs (tRNA, 5s rRNA)

Question 5

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNA polymerases:
all three…

Answer 5

• All three nuclear RNA polymerases across all eukaryotes and bacterial RNA polymerases are monologues
• Two largest units coded on separate genome

Question 6

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure

Answer 6

RNAP II is a multi-subunit enzyme composed of 12 subunits (Rpb1–Rpb12). It’s structurally and functionally conserved across eukaryotes

Catalytic Core (Rpb1 + Rpb2)
Forms a DNA-binding cleft
Active site with Mg²⁺ ions for phosphodiester bond formation
Template DNA enters, RNA-DNA hybrid forms inside the cleft

Cleft and Pore
Channels for:
DNA entry
RNA exit
NTP (nucleotide) entry to the active site

Bridge Helix and Trigger Loop
Highly conserved elements that control RNA synthesis speed and fidelity

Question 7

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
unique features

Answer 7

o CTD
o Terminal domain

Question 7

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
C-Terminal Domain (CTD)

Answer 7

• The largest subunit of RNAP II contains an unsusal C-terminal domain (CTD)
  o The CTD consists of tandem repeats of the consensus sequences
   Tyr-Ser-Pro-Thr-Ser-Pro-Ser
   26, 42, and 52 repeats in yeast, Drosophila and human
• Can predict the complexity of organism based on no. of repeats

Question 8

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
CTD phosphorylation state changes during transcription:

Answer 8

Initiation: Ser5-P

Elongation: Ser2-P

Termination: Dephosphorylation

Question 8

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
C-Terminal Domain (CTD): prior to transcription

Answer 8

o Prior to transcription the CTD is unphosphorylated and is phosphorylated during transcription initiation and elongation
 Serine 2 and serine 5
 Most important modification
 Become phosphorylated
 Important throughput the transcription cycle
 [Initiation -> 5 phosphorlysed (then is de-phosphorylased) —> Elongation -> 2 phosphorylsed ]

Question 9

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
Function–Structure Link

Answer 9

The modular structure allows RNAP II to coordinate transcription with RNA processing (capping, splicing, poly-A addition).

Its dynamic architecture enables promoter escape, elongation, proofreading, and termination.

Question 9

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
Transcription requires…

Answer 9

genetic factors

gene/cell-specific factors

Question 10

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
Transcription requires: genetic factors

Answer 10

o RNA polymerase II
o General (or basal) transcription factors (GTFs)
 TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH
o Mediator
 Large multiprotein complex -> required at all promoters

Question 11

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II structure
Transcription requires: Gene/cell-specific factors

Answer 11

o Activators (& repressors)
o Co-activators (& co-repressors)

Question 12

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
transcription initiation

Answer 12

• For a gene to be transcribed, RNAPII and the GTFs must assemble at the gene to form a pre-initiation complex (PIC)
• On naked DNA (not associated with histones) in vitro, RNAPII and the GTFs form a PIC and will transcribe DNA at low levels
  o Th GTFs and RNAPII may bind sequentially or as large complexes
• Activators are required in a chromatin environment.

Question 13

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
Assembly of the transcription machinery

Answer 13

STEP WISE MODEL
- TFIID - first to bind
o Large protein complex made up of TATA binding protein
- TFIIA
o Not always required
o Believe that in a real cell require TFIIA
- TFIIB
o Single polypeptide chain
- TFIIF
o Delivers RNA polymerase
- Once RNA polymerase is bound
o TFIIE, TFIIH bind
o Initiation

Question 14

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
mediator

Answer 14

• In addition another ubiquitous complex is required – mediator
• A mediator is a large evolutionary conserved compex
• Required at all transcription events

Question 15

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
sequence elements

Answer 15

• Several sequence elements are required to drive transcription
  o Core promoter
   When the PIC assembles
  o Proximal-promoter element
   Binds regulatory proteins
  o Enhancers
   Binds regulatory proteins

Question 16

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
core promoters

Answer 16

• Core promoters can be focused or dispersed
  o Categorised on where transcription starts
• Transcription can start from a no. of points across a (e.g. 100) bp region
• Tend to be expressed all the time (constitutive) –> more difficult to control

Question 17

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
core promoters - focused

Answer 17

o Contain one or more specific sequence elements
 The TATA box, Inr and DPE are recognised by TFIID
 The BRE is recognised by TFIIB
o Typically regulated and can drive high levels of transcription

Question 18

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
core promoters - dispersed

Answer 18

• Most mammalian promoters are dispersed promoters containing CpG islands
  o Regions with relatively high CpG content
• Transcription starts at multiple sites over 100 to 1000 base pairs
• Dispersed core promoters are typically constitutively expressed
  o Housekeeping gene or gene for developmental transcription factors
   A gene that is transcribed most of the time in most cells

Question 19

RNA POLYMERASE AND THE PRE-INITIATION COMPLEX
RNAP II:
core promoters - dispersed
CpG sites

Answer 19

• CpG are sites on the genome that can be methylated
• 5-methyl-cytosine
• Oxidation deamination
• C -> T
  o Lost CpG throughout evolution
  o Expect in places where its hard to methylate (CpG islands -> region of CpG dinucleotide has been maintained, rich compared to rest of genome) or where they are needed for survival

Question 20

ACTIVATION OF TRANSCRIPTION
what is transcribed

Answer 20

• In a eukaryotic cell most genes are not transcribed
  o Nucleosomes will act as general transcriptional repressors

Question 21

ACTIVATION OF TRANSCRIPTION what is required

Answer 21

- Activators are required for transcription in a chromatin environment o For the relief of the repression by nucleosome o And the recruitment of the GTFs and RNAP II to the promoter

Question 22

ACTIVATION OF TRANSCRIPTION where do activators bind

Answer 22

Activator proteins bind to target sequences Recruit co-activators * Open up chromatin structure (loosen) o Add epigenetic tags * Via action of co-activators and mediators lead to recruitment of DNA polymerase II

Question 23

ACTIVATION OF TRANSCRIPTION silent genes?

Answer 23

- Transcriptionally silent genes are present on condensed chromatin containing repression epigenetic modifications (tags)

Question 24

ACTIVATION OF TRANSCRIPTION transcription requires

Answer 24

o The opening of the chromatin  This requires positive epigenetic modifications (tags) o Assembly of the pre-initiation complex

Question 25

ACTIVATION OF TRANSCRIPTION Activation by recruitment:

Answer 25

- Many activators in eukaryotes function by aiding the recruitment of the general transcription factors and RNAP II to the transcription complex

Question 26

ACTIVATION OF TRANSCRIPTION Activation by recruitment: where do activators bind?

Answer 26

- Some activators bind mediator, the GTFs and/or RNAP II o Different activators will recruit different components

Question 27

ACTIVATION OF TRANSCRIPTION Activation by recruitment: activators bind in the...

Answer 27

vicinity of the gene

Question 28

ACTIVATION OF TRANSCRIPTION Activation by recruitment: transcription is controlled by

Answer 28

by several transcription factors

Question 29

ACTIVATION OF TRANSCRIPTION Activation by recruitment: transcription factors

Answer 29

- These activators bind the proximal-promoter element and enhancer sequences and drive assembly of RNAP II and the GTFs at the core promoter

Question 30

ACTIVATION OF TRANSCRIPTION Activation by recruitment: enhancers

Answer 30

- Enhancers work upstream or downstream at relativity long distances away from core promoter o Not required o Transcription can work without it

Question 31

ACTIVATION OF TRANSCRIPTION Enhanceosomes

Answer 31

- Refers to DNA-activator complex - Multiple proteins may binds to form a stereospecific enhancesosome - Co-operative bindng leads to tight regulation - The different activation domains work synergistically

Question 32

ACTIVATION OF TRANSCRIPTION Enhanceosomes - interferon beta

Answer 32

o On/off o Tightly regulated

Question 33

ACTIVATION OF TRANSCRIPTION modular promoters

Answer 33

- Many genes are controlled by multiple enhancers o Allow complex regulation in different cell types, at different time or in response to different stimuli

Question 34

ACTIVATION OF TRANSCRIPTION modular promoters example

Answer 34

E.g. eve gene is turned on in different segments in embryo development

Question 35

ACTIVATION OF TRANSCRIPTION modular promoters - Different enhancers in...

Answer 35

different areas of development / segments o Because different activators bind to different enhancers o So one activator is present in one segment but not in another o Allow for complex regulation  Allows gene to be regulated in different ways in response to e.g. different stimuli - E.g. eve gene is turned on in different segments in embryo development

Question 36

ACTIVATION OF TRANSCRIPTION modular promoters - Enhancers and the tissues they are active

Answer 36

The proximal pituitary enhancer (PPE) - Melanotrophs and corticotrophs of the pituitary Distal pituitary enhancer (DPE) - Pituitary corticotrophs nPE1 and nPE2 - neurons of the hypothalamic arcuate nucleus (Arc)

Question 37

ACTIVATION OF TRANSCRIPTION activator protein structure

Answer 37

- activator proteins have a modular structure

Question 38

ACTIVATION OF TRANSCRIPTION activator protein structure what do they contain

Answer 38

a single DNA binding domain (DBD) at least one activation domain (AD) may contain a regulatory domain - e.g. steroid hormones receptors will contain a ligand binding domain (LBD)

Question 39

ACTIVATION OF TRANSCRIPTION activator protein structure simplistically need...

Answer 39

two functional groups o Binding domain  Typically contain alpha-helix * Fit snugly into major groove of DNA o Activation domain  Sticky patch which allows it to interaction with target protein * Allows it to drive transcription

Question 40

ACTIVATION OF TRANSCRIPTION Activating the activator

Answer 40

- Many activators are present in an inactive state in the cytosol or nucleus - They are activated in response to a signal e.g.

Question 41

ACTIVATION OF TRANSCRIPTION Activating the activator how?

Answer 41

CREB * Goes onto bind co-activators * Leads to transcription activation o External signal binds membrane bound receptor o Which activated Adenylyl cyclase producing cAMP o cAMP activates protein kinase A (PKA) o PKA phosphorylates CREB o CREB binds CRE-element and recruits CBP/p300 which leads to transcription activation

Question 42

ACTIVATION OF TRANSCRIPTION signalling pathways

Answer 42

- The response to many signaling pathways is a change in gene expression patters, e.g. o G protein coupled receptors o Cytokine receptors o Receptor tyrosine kinase o TGFb-receptors o Hedgehog receptors o Wnt receptors o Notch receptor - Involve (at least to some extent) involve activation of transcription factors

Question 43

TRANSCRIPTION REPRESSION nucleosome

Answer 43

- The nucleosome act as a general transcription repressors o Activators are therefore required at all eukaryotic promoters

Question 44

TRANSCRIPTION REPRESSION specific?

Answer 44

- Gene-specific repression of transcription if often employed to provide a diverse set of regulatory patterns

Question 45

TRANSCRIPTION REPRESSION fine tuning?

Answer 45

- Repressor protein fine tuning activator o (in ways a repressor targets the activator rather than the gene)

Question 46

TRANSCRIPTION REPRESSION mechanisms of hindrance

Answer 46

Steric Hindrance Masking Some repressors sequester the activator in the cytoplasm

Question 47

TRANSCRIPTION REPRESSION mechanisms of hindrance: Steric Hindrance

Answer 47

- The repression binds a DNA site that overlaps an activator binding site

Question 48

TRANSCRIPTION REPRESSION mechanisms of hindrance: Masking

Answer 48

- The repressor binds the activator and masks the activation domain o The repressor may or may not bind the DNA - Repression domain that masks activator domain

Question 49

TRANSCRIPTION REPRESSION mechanisms of hindrance: sequester

Answer 49

Some repressors sequester the activator in the cytoplasm

Question 50

EPIGENETICS Transcription in a chromatin environment

Answer 50

- Activators must work in a chromatin context o Transcribed genes are still associated with nucleosomes and higher-order chromatin structure - Activator proteins have to overcome the steric hindrance of the nucleosome o Many activators recruit co-activator that open the chromatin to facilitate transcription

Question 51

EPIGENETICS involves

Answer 51

- DNA methylation, chromatin remodelling and cromatin modification are examples of epigenetic regulation

Question 52

EPIGENETICS o Chromatin remodelling can...

Answer 52

 Can activate or repress transcription

Question 53

EPIGENETICS o DNA methylation is...

Answer 53

 Linked to transcription repression

Question 54

EPIGENETICS o Histone acetylation is...

Answer 54

 Linked to transcription activation

Question 55

EPIGENETICS o Histone methylation is...

Answer 55

 Linked to activation and repression

Question 56

EPIGENETICS plays a role in

Answer 56

- Epigenetics plays an important role in cellular homeostasis, development an disease

Question 57

EPIGENETICS Chromatin remodelling: unmasking core promoter

Answer 57

- Some co-activators (e.g. SWI/SNF) remodel (move) the nucleosomes on the DNA template o This may unmask the core promoter or other important sites

Question 58

EPIGENETICS Chromatin remodelling: what does it move

Answer 58

- Moves relative position of nucleosomes o Away from core promoter o Available for assembly

Question 59

EPIGENETICS Chromatin modification

Answer 59

- Post-translationally modified - Some co-activator covalently modify histone proteins, e.g. acetylation and methylation

Question 60

EPIGENETICS Chromatin modification: Repressors and chromatin structure

Answer 60

- Some transcriptional repressors will recruit corepressors containing histone deacetylases (HDACs) o HDACs deacetylate histones, leading to transcriptional repression

Question 61

EPIGENETICS Chromatin modification HAT activity

Answer 61

- Co activators with histone acetyl transferase activity (HAT) (e.g. GCN5 and CPB/p300), acetylate the N-terminal tails of histones, reducing their affinity for DNA and acting as a mark that is recognised by other transcription factors o Recruits a co-activator complex o Tend to be acetylated at lysine residues (marked by green blobs)  Neutralises +ve charge  Reducing affinity for DNA

Question 62

EPIGENETICS histone methylation

Answer 62

- Lysine residues in the N-terminal tails of the histone proteins can be covalently modified o Mono-, di-, and tri- methylation  Each replacing a H in the amino group - Different methylation marks are positive or negative

Question 63

EPIGENETICS histone methylation what is methylated?

Answer 63

monomethylation of lysine 4 of histone 3 dimethylation of lysine 9 of histone 3 trimethylation of lysine 27 of histone 3

Question 64

EPIGENETICS histone methylation methylation states of lysine

Answer 64

- Still +vely charged - Histone methylation does not change the charge

Question 65

EPIGENETICS histone modification

Answer 65

- Where the histone is relative to DNA template is relevant - Different patterns

Question 66

EPIGENETICS histone modification associated with active and repressed genes: acetylated lysine

Answer 66

sites of modification: H3, H4, H2A, H2B activation

Question 67

EPIGENETICS histone modification associated with active and repressed genes: hypoacetylated lysine

Answer 67

repression

Question 68

EPIGENETICS histone modification associated with active and repressed genes: phosphorylated serine/threonine

Answer 68

sites of modification: H3, H2A, H2B activation

Question 69

EPIGENETICS histone modification associated with active and repressed genes: methylated arginine

Answer 69

sites of modification: H3, H4 activation

Question 70

EPIGENETICS histone modification associated with active and repressed genes: methylated lysine

Answer 70

modification sites: H3, H4 activation, elongation, repression

Question 71

EPIGENETICS histone modification associated with active and repressed genes: ubiquitimethylated lysine

Answer 71

modification sites: H2B, H2A activation, repression

Question 72

EPIGENETICS DNA methylation and repression

Answer 72

- The 5-position of cytosines within CpG dinucleotides can be methylated o CpG: Palindromic sequence - During differentiation, genes are marked as being “inactive” by methylation o Genes with the potential to be transcribed are not methylation - DNA methylation can lead to transcriptional repression via several possible mechanisms

Question 73

EPIGENETICS DNA methylation and repression: can directly block...

Answer 73

- DNA methylation may directly block the binding of transcription factors - Methyl-CpG binding protein (e.g. MeCP2) recruit HDACs o Histones are deacetylated and other repressors bind leading to the formation of heterochromatin

Question 74

EPIGENETICS DNA methylation and repression: X-inactivation

Answer 74

- In female mammals one X-chromosome is inactivated in each cell - Inactivated is due to DNA metylation o DNA methylation directs the recruitment of HDACs - The nucleosome becomes deacetylated which leads to the formation of condensed chromatin and gene silencing

Question 75

EPIGENETICS DNA methylation and repression: aberrant DNA methylation (aging)

Answer 75

- Aging o As we age methylation patterns are not maintained and this results in global hypomethylation o Activate and passive DNA methylation as cells divide  As we age methylation patterns decay (slowly - e.g. 80-90%)  Epigenetic clocks  Relatively predictable

Question 76

EPIGENETICS DNA methylation and repression: aberrant DNA methylation (cancer)

Answer 76

o Global hypomethylation (Global loss of DNA methylation) occurs in cancer o However there is often hypermethylation (Genes methylated as part of cancer development) of specific CpGs e.g. leasing to silencing of tumour suppressor genes o Epigenetics play an important role in cancer  Can get uncontrolled growth by messing with epigenetics * Artificial * Therapeutic target?

Question 77

EPIGENETICS stress and aging

Answer 77

- Increased epigenetic aging can increase risk of developing a number of diseases - Some of these epigenetic processes can be reversed o E.g. changing lifestyle * Increase epigenetic resistance o Has been shown that psychotherapy can reverse these epigenetic processes * Small amount of studies but results are consistent * Soldiers + PTSD remission saw epigenetics return to pre-service

Question 78

EPIGENETICS Cell-type specific activation

Answer 78

- The same signalling pathways and hence transcription factors can activate or repress different sets of genes in different cell types

Question 79

EPIGENETICS Cell-type specific activation: - For an activator to turn on a gene:

Answer 79

o The chromatin needs to be in a more open conformations (e.g. the DNA is not methylated)  Doesn’t matter how many activators, Transcriptionally silenced o Partner activators need to be present Example in diagram o Green activator once phosphorylated binds cooperatively with red and/or blue activators to turn on the gene present in more open chromatin in that cell type o Binding sites for green, red or blue activators in closed chromatin are unavailable and hence associated genes remain off in that cell type

Question 80

EPIGENETICS A model for transcription initiation

Answer 80

- Pioneer activators bind and recruit chromatin modifying and remodelling complexes o Chromatin is modified (e.g. histone acetylation) and remodelled - Further activator may bind o The activators then recruit the transcription machinery - A pre-initiation complex is formed o RNAP II initiates transcription

Question 81

EXAMPLE PROMOTERS Transcription of the yeast HO gene: Where does the activator bind?

Answer 81

- The activator, SWI5, binds the enhancer 1200-1400 bp away from the transcription start

Question 82

EXAMPLE PROMOTERS Transcription of the yeast HO gene: what does SWI5 recruit

Answer 82

- SWI5 recruits SWI/SNF o SWI/SNF remodels the chromatin in an ATP dependent manner - SWI5 recruits GCN5 o GCN5 has HAT activity which acetylates the histones - The DNA template is now more accessible for other activators and for the transcription machinery, it unpacks the chromatin

Question 83

EXAMPLE PROMOTERS Transcription of the yeast HO gene: SBF activator

Answer 83

- The activator SBF now binds the accessible proximal-promoter region - SBF recruits mediator - Mediator then directs the assembly of the pre-initiation complex

Question 84

EXAMPLE PROMOTERS Transcription of the Interferon Beta gene:

Answer 84

- Upon virus infection, the activators bind the enhancer - A co-activator with HAT activity binds transiently and acetylates the histones - RNAP II, bound to another co-activator is recruited but does not bind the core promoter - A chromatin remodelling complex binds - The nucleosome that is blocking the core promoter is moved - A pre-initiation complex is formed and transcription initiates

Question 85

EXAMPLE PROMOTERS IFN-beta and coronavirus infection: role of IFN-beta

Answer 85

- For all viruses, expression of IFN-beta plays a major role in the host defense against coronavirus infection

Question 86

EXAMPLE PROMOTERS IFN-beta and coronavirus infection: what does recognition activate?

Answer 86

- Host receptor proteins recognise the virus, this leads to activation of IFN-Beta and other pathways to tackle viral infection

Question 87

EXAMPLE PROMOTERS IFN-beta and coronavirus infection: invasion of immune system

Answer 87

- Coronaviruses evade detection by the innate immune system and inhibit interferon production

Question 88

EXAMPLE PROMOTERS IFN-beta and coronavirus infection: timing of interferon response correlated with...

Answer 88

disease severity

Question 89

EXAMPLE PROMOTERS IFN-beta and coronavirus infection: - Coronavirus proteins block...

Answer 89

IFN-beta production e.g. block activation of the activator proteins required for IFN-beta transcription

Question 90

EXAMPLE PROMOTERS Transcription in development and evolution

Answer 90

- The difference between human and other animals is not predominantly due to different genes but how the same set of genes is regulated - E.g. o Prodynorphin  Involved in learning, memory, experience of pain and social attachment and bonding o Humans produce more prodynorphin

Question 91

EXAMPLE PROMOTERS What makes us human

Answer 91

- Increased transcription of the prodynorphin gene is found in humans compared to other primates and mammals o This is due to sequence change sin an enhancer sequence o Enhancer has evolved in humans by picking up 5 mutations

Question 92

EXAMPLE PROMOTERS Gene regulation and disease

Answer 92

- Deregulation of gene expression often leads to disease - There can be different molecular causes - E.g. o Transcription factor mutation o Response element (enhancer) mutation o Aberrant chromatin modification or remodelling o Altered gene dosage leads to an imbalance in the control o Error in mRNA processing

Question 93

EXAMPLE PROMOTERS Histone acetylation – memory and disease

Answer 93

- Histone acetylation plays an important role in neurodegenerative diseases and memory

Question 94

EXAMPLE PROMOTERS Histone acetylation – memory and disease - Hypoacetylation

Answer 94

- Hypoacetylation is found in neurodegenerative disease o Inhibition of HDAC activity may provide protection o Rubinstein-Taybi syndrome is caused by mutations in CBP/p300  Results in loss of HAT activity (Mutation within histone acetyl transferase) leading to problems with cognition

Question 95

EXAMPLE PROMOTERS Histone acetylation – memory and disease - Histone acetylation plays an important role in...

Answer 95

memory o HDAC inhibitors improve memory

Question 96

EXAMPLE PROMOTERS Gene regulation and cancer: TP53

Answer 96

inactivation of the tumour supressor gene TP53 is observed in most human cancers p53 is a transcription factor

Question 97

EXAMPLE PROMOTERS Gene regulation and cancer: promoters and enhancer mutations can...

Answer 97

cause cancer

Question 98

EXAMPLE PROMOTERS Gene regulation and cancer: deregulation of histone remodelling plays a role in...

Answer 98

cancer e.g. increased nucleosome density around the promoter suppressor gene

Question 99

EXAMPLE PROMOTERS Gene regulation and cancer: deregulation of epigenetics

Answer 99

deregulation of epigenetic processes plays a key role in cancer development and progression

Question 100

EXAMPLE PROMOTERS Gene regulation and cancer: p53

Answer 100

- P53 - mutations linked to many human cancers o P53 itself o Or an activity involving p53 tumour suppression has been mutated

Question 101

POST TRANSCRIPTIONAL CONTROL

Answer 101

- RNA processing and alternative splicing - Export of mRNA from nucleus to cytoplasm - Translation control - mRNA stability - protein stability

Question 102

POST TRANSCRIPTIONAL CONTROL - Translation control

Answer 102

o Translation initiation factors (e.g. eIF2) can be regulated altering global transcription o Some mRNAs contain specific sequences in their 5’-UTR which bind proteins that control translation  E.g. ferritin mRNA

Question 103

POST TRANSCRIPTIONAL CONTROL transcription and pre-mRNA processing:

Answer 103

- the pre-mRNA is processed during transcription to give a mature message that can be translated o As RNA being made introns removed and exons being stuck together o 5' cap important for ribosome binding (for translation) and stability of the mRNA  Poly A tail

Question 104

POST TRANSCRIPTIONAL CONTROL transcription and pre-mRNA processing: Alternative splicing

Answer 104

- The same pre-mRNA can be alternatively spliced to give slightly different versions of the same protein - Approx. 95% of human genes are alternatively spliced - Alternative splicing is responsible for the relative morphological and physiological complexity of mammals o High eukaryotes  Don’t contain more genes  Just a higher level of alternative splicing

Question 105

POST TRANSCRIPTIONAL CONTROL Mis-splicing and neurodegeneration

Answer 105

- Mis-splicing contributes to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) - The protein TDP-43 is normally found in the nucleus and plays an important role in splicing of UNC13A - In ALS and FTD, TDP-43 aggregates in the cytoplasm o UNC13A is mis-spliced and ultimately degraded o The loss of UNC13A protein is linked to neurodegeneration

Question 106

POST TRANSCRIPTIONAL CONTROL Mis-splicing and neurodegeneration : Proteins factors that control alternative splicing

Answer 106

o Incorrect splicing o --> loss of protein product because of degradation

Question 107

POST TRANSCRIPTIONAL CONTROL Translation control in response to Fe:

Answer 107

- Ferritin is an iron storage protein required when iron level are high - The 5’-UTR of the ferritin mRNA contains an iron-response element (IRE) o Low iron levels lead to recruitment of the IRE-binding protein which blocks translation o Iron binds the IRE-binding protein and stops it binding the 5’-UTR

Question 108

POST TRANSCRIPTIONAL CONTROL Translation control in response to Fe: Iron binds the IRE-binding protein

Answer 108

Still stable but ribosome cannot interact * Iron blocking site on IRE-binding protein * Cannot bind to 5'-UTR * No iron translated

Question 109

POST TRANSCRIPTIONAL CONTROL Translation control in response to Fe: mRNA stability

Answer 109

- mRNA half life varies - stability is proportional to length of the polyA tail

Question 110

POST TRANSCRIPTIONAL CONTROL Translation control in response to Fe: mRNA stability: 3'-UTR

Answer 110

- 3’-UTR may contain sequences that affect stability o E.g. IRE sequence in 3’-UTR of the mRNA for the transferrin receptor protein  Transferrin is the reverse of ferritin o Binding of the IRE-binding protein stabilises the mRNA

Question 111

POST TRANSCRIPTIONAL CONTROL Translation control in response to Fe: IRE binds and stops it from being....

Answer 111

degraded - (in a low iron environment -> increased levels of protein) - When iron levels increase o Fe binds to IRE-binding protein o Conformational changes -> release from 3'-UTR o 3'-UTR exposed -> can bind and be degraded

Question 112

POST TRANSCRIPTIONAL CONTROL RNA interference (RNAi) – siRNAs

Answer 112

- Small interfering RNAs (siRNAs) are typically generated from exogenous dsRNA by Dicer o 21-23 bp with 2 nucleotide 3’ overhang - They bind te RISC complex to form siRISC - One RNA strand is degraded - The argonaute protein within the siRISC complex degrades complementary mRNA

Question 113

POST TRANSCRIPTIONAL CONTROL RNA interference (RNAi) – siRNAs steps:

Answer 113

1. dicer cleaves DS RNA into an siRNA about 21-22 bp 2. the siRNA is combined with RISC proteins 3. one of the two RNA strands is degraded 4a. srRNA binds to mRBA 4b. siRISC enters nucleus and binds DNA

Question 114

POST TRANSCRIPTIONAL CONTROL RNA interference (RNAi) is a...

Answer 114

natural process that turns off genes by using a gene's DNA sequence

Question 115

POST TRANSCRIPTIONAL CONTROL RNA interference: STEPS

Answer 115

1. Double-stranded RNA (dsRNA) triggers RNAi. 2. The enzyme Dicer cuts dsRNA into small fragments called small interfering RNAs (siRNAs). 3. The siRNAs silence genes with homology to the double strand.

Question 116

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs

Answer 116

- microRNAs (miRNA) are generated from a larger RNAs (pri-miRNAs) generated by endogenous transcription - the pri-miRNA is processed by Drosa and then Dicer producing 19-25 nucleotides miRNA - the miRNA binds the RISC complex to form mrRISC - the miRISC complex can target mRNA for several genes inhibiting translation

Question 117

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs what are they

Answer 117

MicroRNAs (miRNAs) are small, noncoding RNAs that are part of RNA interference (RNAi), a gene-silencing mechanism Generated by endogenous transcription (Coded for within the genome of the organism)

Question 118

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs drosha

Answer 118

protein complex * Processes the pri-microRNA to produce pre-miRNA

Question 119

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs dicer

Answer 119

exports it and produces mature miRNA * A single stranded RNA moleciles miRNA now binds RISC (miRISC complex)

Question 120

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs miRISC complex

Answer 120

miRNA now binds RISC (miRISC complex) miRISC complex can now target mRNA and inhibit translation * Multiple targets

Question 121

POST TRANSCRIPTIONAL CONTROL RNA INTERFERANCE: miRNAs STEPS

Answer 121

1. transcription 2. cleavage (drosha), pre-miRNA 3. export and cleavage (Dicer), miRNA 4. proteins added, miRISC 5a. partly complementary to messenger RNA - targeted messenger RNAs - inhibition of translation

Question 122

POST TRANSCRIPTIONAL CONTROL How do miRNAs compare to siRNAs?

Answer 122

* Small interfering RNAs (siRNAs) are derived from long double-stranded RNAs and are often exogenous * siRNAs are highly specific with only one mRNA target, whereas miRNAs have multiple targets

Question 123

POST TRANSCRIPTIONAL CONTROL Translational and RNAi - Therapies

Answer 123

* New gene therapies * To treat high cholesterol (when statins are not working)

Question 124

RNA POLYMERASES RNA polymerase I transcription

Answer 124

- In the typical cell, ribosomal RNA accounts for 50% of nascent RNA synthesis - Eukaryotes contain hundreds of copies of the rRNA operons present in the nucleolar organising regions - Each gene being transcribed by multiple RNA polymerase I molecules

Question 125

RNA POLYMERASES RNA polymerase I transcription arranged?

Answer 125

- Tandemly arranged at different locations

Question 126

RNA POLYMERASES RNA polymerase I transcription the intergenic sequence

Answer 126

- The intergenic sequence (IGS) contains terminator sequences (T and To), a spacer promoter (SP) and the rRNA promoter (P)

Question 127

RNA POLYMERASES RNA polymerase I transcription IGS: rRNA promoter

Answer 127

- The rRNA promoter o Core element -45 to +18 o Upstream control element -156 to -107

Question 128

RNA POLYMERASES Transcription initiation by RNAP I 1. UBF

Answer 128

- UBF binds the UCE and SL1 binds the core promoter o SL1 is a multiprotein complex including TBP and TAFs

Question 129

RNA POLYMERASES Transcription initiation by RNAP I 2. RNAP I and RRN3

Answer 129

- RNAP I and RRN3 are recruited o The nRRN3 complex delivered RNAP I to SL1

Question 130

RNA POLYMERASES Transcription initiation by RNAP I 3. initiation

Answer 130

- After several abortive attempts, transcription initiates o hRRN3 is released, UBF and SL1 remain bound and act as a scaffold for reinitiation

Question 131

RNA POLYMERASES Transcription initiation by RNAP I 4. termination

Answer 131

- termination occurs when RNAP I reaches TTF-1 and PTRF

Question 132

RNA POLYMERASES Regulation of RNAP I transcription

Answer 132

- RNAP I transcription is regulated in response to several factors including the cell cycle - Regulatory mechanisms include: o Chromatin structure o Modification  E.g. phosphorylation of the different transcription factors

Question 133

RNA POLYMERASES RNA polymerase III: type 1 promoters

Answer 133

- Type 1 promoters recruit RNAP III, TFIIIA, TFIIIC and a TFIIIB complex that contains BFR 1, BDP 1, and TBP

Question 134

RNA POLYMERASES RNA polymerase III: type 2 promoters

Answer 134

- Type 2 promoter recruit RNAP III, TFIIIC, and a TFIIIB complex that contains BRF 1, BDP 1, TBP

Question 135

RNA POLYMERASES RNA polymerase III: type 3 promoters

Answer 135

- Type 3 promoters recruit RNAP III, SNAPc (Binds to an upstream response element), and a TFIIIB complex that contains BRF 2, BDP 1, and TBP

Question 136

RNA POLYMERASES RNA polymerase III: protein factors

Answer 136

there are different protein factors

Question 137

RNA POLYMERASES RNA polymerase III: regulation

Answer 137

- RNAP III activity is regulated in response to many signals including the cell cycle, differentiation, and cell stress

Question 138

RNA POLYMERASES RNA polymerase III: - Mutations in the RNAP III machinery can give rise to...

Answer 138

a number of diseases

Question 139

RNA POLYMERASES RNA polymerase III: Mutations in the RNAP III machinery: RNAP III

Answer 139

 Leukodystrophies (neurodegenerative diseases)  Immunodeficiency

Question 140

RNA POLYMERASES RNA polymerase III: Mutations in the RNAP III machinery: BFR 1

Answer 140

 Cerebellar-facial-dental syndrome  Cancer

Question 141

RNA POLYMERASES RNA polymerase III: Mutations in the RNAP III machinery: BFR 2 and TFIIIC

Answer 141

cancer

Question 142

RNA POLYMERASES RNA polymerase III: Mutations in the RNAP III machinery: BDP I

Answer 142

 Hereditary hearing loss

Question 143

WEAKE WORKMAN REVIEW: key mechanims of transcription in euks:

Answer 143

1. Activator-dependent recruitment 2. Chromatin Remodeling: 3. Transcription Elongation and Pausing

Question 144

WEAKE WORKMAN REVIEW: key mechanims of transcription in euks: 1. Activator-dependent recruitment

Answer 144

Transcription factors bind to specific DNA sequences and recruit co-activators, such as the SAGA and Mediator complexes, which are essential for the formation of the pre-initiation complex (PIC) and the recruitment of RNA polymerase II (Pol II) .

Question 145

WEAKE WORKMAN REVIEW: key mechanims of transcription in euks: 2. Chromatin Remodeling:

Answer 145

Chromatin must be made accessible for transcription, a process that involves nucleosome remodeling complexes like SWI/SNF, which are recruited by transcriptional activators. These complexes help in repositioning or displacing nucleosomes, facilitating the assembly of the transcription machinery .

Question 146

WEAKE WORKMAN REVIEW: key mechanims of transcription in euks: 3. Transcription Elongation and Pausing

Answer 146

After the formation of the PIC, Pol II can stall at promoter-proximal sites before elongation. This pausing is a common feature in inducible genes like those in heat-shock responses . For some genes, such as the Gal genes, the release of paused polymerase into productive transcription elongation requires further signaling and co-activator interactions

Question 147

WEAKE WORKMAN REVIEW: the yeast HO gene regulation with Gal4-mediated induction of Gal gene expression: * Gal4-mediated induction:

Answer 147

In the Gal genes, the transcriptional activator Gal4 binds to upstream activating sequences (UAS) and recruits co-activators like SAGA and Mediator . The repression of Gal4 is relieved by Gal3, which sequesters the Gal80 repressor in the presence of galactose, allowing transcriptional activation .

Question 148

WEAKE WORKMAN REVIEW: the yeast HO gene regulation with Gal4-mediated induction of Gal gene expression: * HO gene regulation

Answer 148

The HO gene in yeast is regulated by a different set of activators, primarily through the Swi5 and Ace2 transcription factors, which control its expression during specific cell cycle stages . HO expression is tightly regulated, and the recruitment of co-activators and the chromatin remodeler SWI/SNF are essential for proper gene activation. Unlike the Gal genes, which are subject to a clear activation mechanism by Gal4, HO regulation also includes specific interactions with chromatin and cell cycle regulators .

Question 149

WEAKE WORKMAN REVIEW: conclusions:

Answer 149

, while both the Gal genes and the HO gene rely on activator-dependent recruitment and chromatin remodeling, the specifics of their regulation, including the role of repressors and the timing of activation, differ. Gal genes are primarily controlled through the interaction of Gal4 with Gal80, whereas HO gene regulation is more complex, involving cell cycle-dependent activators like Swi5 and Ace2.

Question 150

RNA Polymerase RNAP I chatgpt

Answer 150

Transcripts: rRNA (except 5S) Promoter Elements: UCE, Core General TFs: UBF, SL1 Regulation Notes: Regulated via chromatin & phosphorylation

Question 151

RNA Polymerase RNAP II chatgpt

Answer 151

Transcripts: mRNA, miRNA, snRNA Promoter Elements: TATA, Inr, DPE General TFs: TFIID, etc. Regulation Notes: Regulated via CTD, GTFs, Mediator

Question 152

RNA Polymerase RNAP III chatgpt

Answer 152

Transcripts: tRNA, 5S rRNA Promoter Elements: Internal (type I/II/III) General TFs: TFIIIA-C, BRF1/2 Regulation Notes: Activity linked to cell cycle/stress

Question 153

role of the core promoter, RNAP II, general TFs, and Mediator: mediator serves as a... chatgpt

Answer 153

bridge between activators bound at enhancers and the core transcription machinery.

Question 154

Chromatin context, activators & co-activators pioneer transcription factors chatgpt

Answer 154

(e.g., FOXA1) that can bind closed chromatin and recruit remodelers.

Question 155

Chromatin context, activators & co-activators flow summary chatgpt

Answer 155

Activator binds → recruits co-activator (HAT/SWI-SNF) → chromatin opens → Mediator & PIC assemble → transcription begins

Question 156

Post-transcriptional control real-world example of RNAi-based therapy chatgpt

Answer 156

Inclisiran for cholesterol control

Question 157

Post-transcriptional control schematic timeline: chatgpt

Answer 157

Pre-mRNA → Splicing → capping/poly-A → nuclear export → translation control → degradation

Question 158

Role of transcription in evolution and disease eQTLs chatgpt

Answer 158

(expression quantitative trait loci) and how variation in regulatory regions contributes to evolution and disease. Tie it into how transcriptional misregulation can lead to different diseases based on the type and location of error (TF mutation vs enhancer vs epigenetic change).