Control of eukaryotic gene expression - CMB2001

What is transcription defined as

Transfer of genetic info from dsDNA into ssRNA (mRNA)

What are the 4 different nucleotide triphosphotases that are used in transcription

ATP

UTP

CTP

GTP

How does transcription work in prokaryotes (bacteria)

RNA polymerase + sigma factor (creating a holoenzyme) will bind upstream of the promoter (closed complex), it will then melt DNA strands acting as a helicase allowing for mRNA formation (open complex)

Where are the 2 locations promoters are located in prokaryotes

-35 and -10

What is a consensus sequence

a sequence of DNA having similar structure and function in different organisms (eg TATA box)

What are the 2 regions of eukaryotic promoters

Regulatory region and core (basal) region

What is the methylation of CpG islands associated with

silencing of transcription

What is the main difference between transcription in eukaryotes and transcription in prokaryotes

. Initiation occurs at a lower rate and at several start points in eukaryotes - more complicated

What are Upstream activator sites (UAS) and enhancers

Activator binding sites

What are upstream respressor sites and silencers

Repressor binding sites

What are 2 tools used for identifying promoter elements

. Sequence comparison (consensus seq)

. Reporter analysis

What is the structural difference between prokaryotic RNA polymerase compared to eukaryotic RNA polymerase

. Structurally similar with clear homologues of certain subunits, however 12 sub in euk and 5 in pro (B, B', a, a and w)

What are general transcription factors (GTFs)

transcription factors that are required for transcription initiation by RNA polymerase (ie TFIID, TFIIH)

What is the structure of the pre initiation complex (PIC)

. TFIID, IIA, IIB, IIF, RNA pol II and IIE, IIH

What is the C terminal domain (CTD) and what happens to it as polymerase II begins transcribing

A series of repeats located at the c terminal end of the largest subunit of RNApol II (B' homologous subunit), it is extensively phosphorylated as polymerase begins transcribing

What does TFIIH contain that plays a key role in promoter melting

ATPase called XPB (or Ss12), it pushes DNA into the cleft where RNA polymerisation is catalysed

What are the 3 major classes of RNA polymerases in eukaryotes and what do they do

RNApol 1, 2 and 3

. 1 - synthesises most rRNA (nucleolus)

. 2 - synthesises mRNA, snRNA and miRNa (nucleoplasm)

. 3 - synthesises tRNA, 5S rRNA (nucleoplasm)

What is the anagram to remember the assembly of the PIC

Dick And Balls For POLly, EH?

What is the activity like of basal transcription vs activated transcription

Inactive/low, activated has enhancers so transcription is a lot higher

What are the 2 different types of enhancers

. Common sequence elements

. Response elements

What are common sequence elements (3)

Those that are located close to core promoter and tend to be continuously active, binding activators that are relatively abundant in cell

What are response elements

Those that bind factors who's activity is controlled in response to specific stimuli (ie heat shock and growth factors)

What is a brief overview of combinatorial control of transcription (using heat shock and growth factors as an example)

The type and combination of elements dictate when and at what level a gene is transcribed

How are activators that are bound to enhancer elements brought into contact w basal transcription machinery

By looping out of the intervening DNA

What are activator proteins usually described as and what are they typically composed of

Modular - composed of multiple units

. DNA binding domain and one or more activation domains

DNA binding domains have unique 3D structure, what are 4 examples?

. Leucine zipper

. Zinc finger

. Homeodomain

. Helix loop helix

What is the structure of activation domains like and how are they often characterised (3 examples?)

. Often unstructured, characterised by amino acid composition

. Acidic patch, glutamine rich and proline rich

What are the 4 main ways that activators work

. Promotion of additional activator binding

. Stimulation of PIC assembly (recruitment)

. Releasing stalled RNA polymerase (stimulate activity

. Modulation of chromatin

What components of the PIC complex do activators interact with to promote assembly (3)

. TFIID

. TFIIB

. Mediator

What is the mediator complex?

A complex of proteins that interacts with the Pol II complex and allows transcription to begin.

. It acts as a bridge between DNA binding transcription factors and RNA pol II

What is the repeating unit of chromatin

Nucleosome

What is the structure/composition of nucleosomes

Composed of an octamer of core his tones associated w DNA

What are core histones and give 4 examples

. Small highly conserved basic proteins w flexible N-terminal tail and a globular C-terminal domain

. H2A, H2B, H3 and H4

What are the 2 basic types of histones

core histones and linker histones

What are histone variants

. Distinct proteins that replace canonical histones in nucleosomes altering the property of the nucleosome

. They are coded for by genes that differ from the highly conserved major types

What are 2 of the main post-translational modifications that histones are subjected to

. Acetylation and methylation

Where does post-translational modification usually take place on histones

N-terminal tails

How is acetylation mediated in regards to histones PTM

Histone acetylases (HATs)

Histone deacetylases (HDACs)

How does acetylation mediated transcriptional activation (2)

. Alters the charge on the histone rail, so DNA not as tightly bound to histones

. Directs the recruitment of bromodomain proteins

What are bromodomain proteins

Reader modules that bind to acetylated lysines, usually promote transcription by acetylating nucleosomes (loosens chromatin)

Where's does histone methylation usually occur and by what

On lysine residues

. By histone lysine methyl transferases (HKMTs) and lysine demethylases

What impact does histone lysine methylation have on transcription

. Can be either positive or negative, its function is context dependant

How do ATP-dependant chromatin remodellers work

Use ATP to reposition the nucleosome

What do Snf2-related ATPases do/work

They are similar to helicases but instead of unwinding DNA they translocate along DNA and shift histones

What are rage 5 different types of ATP-dependant chromatin remodelling

. Sliding

. Unwrapping

. Eviction (removes nucleosome)

. Spacing

. His tone variant exchange

How does SWI/SNF remodel chromatin and how does this aid in transcription

. It's motor activity generates torsional stress weakening his tone-DNA interactions, this can expose promoter regions for transcription factors

What are the 3 distinct mSWI/SNF complexes

. Canonical BAF (cBAF)

. Polybromo-associated BAF (PBAF)

. Noncanonical BAF (ncBAF)

What are the mSWI/SNF complexes essential for (3)

Regulating gene expression, cell differentiation and DNA repair

What disorders are associated with mutations in genes encoding SWI/SNF subunits

Neurodevelopmental disorders (cancers)

Chromatin structure is often exploited to cause what

Transcriptional repression/activation

What are hyperacetylated regions of the genome associated with

Active regions

What can the NuRD complex (belonging to the Mi2/CHD family) do in terms of nucleosomes

It can slide nucleosomes into repressive positions and remove acetylation marks, thus suppressing transcription (often of tumour suppressor genes)

What are 2 basic types of chromatin and what are there characteristics

. Euchromatin (open and active, gene rich, potential to be transcribed)

. Heterochromatin (transcriptionally silent, compact, gene poor, repetitive regions)

What are 3 biochemical features of heterochromatin

What are the 2 major roles that HP1 does

. Structural (compacts chromatin)

. Recruitment (attracts other repressors) - creates an environment hostile to transcription, keeping heterochromatin silent

What is HP1

heterochromatin protein 1

What is a co-repressor

a molecule that cooperates with a repressor protein to switch an operon off

How is H3 K9me-dependent heterochromatin (H3Lys9me) formed

K9 that's attached to H3 is deacetylated by HDACS

. Methyltransferases (eg Suvar39) they methylate K9

. HP1 then binds to methylated K9, creating a reinforcing loop recruiting more transferases and spreading heterochromatin

How does X-chromosome inactivation occur

through heterochromatin formation

What transcription factor family is central to immune, inflammatory and stress responses

NF-kBs

What is the most important domain in NF-kBs

Rel Homology Domain (RHD), it encodes the DNA binding and dimerisation function of NF-kBs

What are 4 examples of NF-kBs

Rel A (p65)

Rel B

P50

P52

What is ubiquitin

Protein put on intermediate filaments of the cytoskeleton to mark them for degradation in ubiquitin-proteosome degradiation (decrease cell size)

NF-kBs are activated by various stimuli, what is the activation path once stimulated

. upon stim, IkB kinase complex phosphorylates IkB (has NF-kB bound)

. This causes IkB to be ubiquinated, thus allowing the bound NF-kB to freely translocate into nucleus.

What are the 3 core subunits of IKK (IkB kinase complex)

IKKa, Ikkb and IKKy

Where js NF-kB usually found in cell when inactive

In the cytoplasm, bound to IkB

What is the difference between canonical and non-canonical NF-kB pathways (3 each)

. Canonical - rapid, triggered by cytokines, uses IKKbeta -> p65/p50 dimers

. Non-canonical - slower, developmental, relies on NIK and IKKalpha -> RelB/p52 dimers

Which NF-kB dimers typically enter nucleus in non-canonical pathway

RelB/p52

How is No-kB activity regulated by negative feedback

. NF-kB activates transcription of IkBalpha, which then binds NF-kB and re-sequesters it in the cytoplasm

How does NF-kB link innate and adaptive immunity

By inducing cytokine expression that recruits and activates immune cells

What are 3 acute responses to hypoxia

. Hyperventilation

. Tachycardia

. Mild respiratory alkalosis

What is angiogenesis

formation of new blood vessels

What are examples of cellular responses to hypoxia

. Translational block

. Turning off of transcription

. Cell death

. Basically just turning off high energy consuming processes

What is HIF (hypoxia inducible factor) system

Heterodimeric (HIF-a and HIF-1b) transcription factor with the ability to activate hypoxia responsive genes

What happens to HIF under hypoxic conditions

HIF-alpha is not degraded, dimerises with HIF-beta and makes its way into the nucleus where it activates transcription of hypoxic responsive genes

How are HIF-alpha levels controlled under normoxia

PHDs hydroxylate HIF-a, this causes ubiquitination (degradation) of protein preventing unnecessary hypoxic responses

What is an example of hypoxia-responsive genes

VEGF (angiogenesis), erythropoietin (RBC production), or glycolysis enzymes

What is the role of VHL in HIF regulation

VHL is part of the ubiquitin ligase complex that binds hydroxylate HIF-alpha and targets it for degradation

What are PHD (prolyl hydroxylase domain) enzymes and what do they do

. Act as oxygen sensors

. Ass hydroxyl groups to proline to forms hydroxyproline which regulates HIF

What is FIH (factor inhibiting HIF)

An oxygen-dependant enzyme that hydroxylates an asparagine residue in the C-terminal activation domain of a HIF-alpha

In hypoxic conditions how does FIH work

FIH activity is reduced (as it needs oxygen as a substrate), HIF-alpha remains unmodded. HIF-alpha binds CBP/p300 causing active transcription of hypoxia-responsive genes.

In normoxic conditions, how does FIH work

FIH uses oxygen to hydroxylate HIF-alpha, this prevents HIF from binding to transcriptional co-activators, no genes transcribed.

What is p53 and how does it work

Tumour suppressor gene, prevents the propagation of damaged DNA by inducing cell cycle arrest, DNA repair or apoptosis

What protein normally inhibits p53 and how

Mdm2

. An E3 ubiquitin enzyme, binds to p53 ubiquitinating it and targets it for proteasomal degradation

How does DNA damage activate p53

DNA damage -> ATM/ATR kinases phosphorylase p53 -> disrupts Mdm2 binding -> p53 stabilises and accumulates which activates transcription of target genes

What is the Mdm2 feedback loop?

p53 can also stimulate Mdm2 gene expression which forms a negative feedback loop that limits the extent of p53 activation.

What are the main steps to pre-mRNA processing before it can be used in transcription

5' capping, splicing and 3' poly A tail

What are the important conserved sequences found in introns

5' GU, branch point and 3' AG, The spliceosome recognises these conserved sequences at intron boundaries

What is a spliceosome

Large RNA-protein complex that catalyses splicing

What are spliceosome composed of

Made of snRNPs (small nuclear ribonucleoproteins), U1,2,4,5 and 6

Can you describe / explain how introns are removed using a spliceosome, including the different components of said spliceosome

. U1 binds the 5' splice site

. U2 binds the branch point (an A nucleotide)

. U4/U6-U5 complex joints and rearrangement occurs

. Introns removed as a lariat loop structure

. Exons ligase together

What is alternative splicing?

mRNA processing events that lead to different combinations of exons being spliced together

What does alternative splicing result in

generation of different isoforms from a single gene

How is alternative splicing regulated

can be regulated by Repressors that bind to Exon or Intron Splicing Silencers (ESS or ISS) and by Activators call SR proteins that bind to Exon or Intron Spicing Enhancers (ESE or ISE)

Splicing is an RNA catalysed event.

What is nascent RNA?

new RNA that is still attached to poly 2

What positioning is conserved sequences AAUAAA located at in regard to PAS

10-35 nucleotides upstream of poly A site (PAS)

What is the enzyme that catalyses addition of poly A tail

Poly A polymerase (PAP)

What is the poly A site and what is it known as

Known as PAS (polyadenylation signal) and it is where pre-mRNA is cut from transcription machinery

What does USE stand for (polyadenylation)

U-rich upstream element