Lecture 3 - Gene Expression and Regulation

Question 1

Chromatin architecture and gene expression

Answer 1

Chromatin structure must be unwound to allow transcription factors and other regulatory proteins access to DNA.

Question 2

How is chromatin structure controlled?

Answer 2

Histone modifications

DNA methylation

Question 3

Histone modifications

Answer 3

Modifications to histone proteins determine how tightly the DNA is coiled.

There are several types of modifications including phosporylation, methylation and acetylation.

Modifications are added and removed by a series of enzymes.

Modifications can open up DNA (H3K9ac, H4K5ac, H3K14ac) or close it (H3K9me, H3K27me).

Question 4

DNA methylation

Answer 4

DNA can also be methylated.

Cytosine residues in the DNA are the usual site of methylation, particularly where they ar enext to a guanine (CpG).

DNA sequences enriched in CpGs are called CpG islands.

The presence of methylation usually (but not always) switches genes off.

Question 5

Nuclear organisation and chromosome territories

Answer 5

Different chromosomes occupy different ‘territories’ in the nucleus and this can alter depending on the activity of genes contained on them.

Chromosomes that contain genes that are co-regulated tend to co-localise within the nucleus.

Question 6

RNA is transcribed by RNA polymerases

Answer 6

RNA polymerase I – transcribes rRNA
RNA polymerase II – transcribes mRNA
RNA polymerase III – transcribes tRNA & snRNA

RNA polymerase II

12 subunits, 550 kDa
Largest subunit contains C terminal domain (CTD) highly phosphorylated during initiation.
Interacts with transcription factors.

Question 7

Transcription Initiation

Answer 7

Transcription is regulated by specific proteins (transcription factors) binding to specific sequences (promoters or enhancers).

Question 8

Core promoter sequences

Answer 8

GC box – GGGCGG
CAAT Box - CCAAT
BRE box – (G/C)(G/C)(G/A)CGCC
TATA box - TATA(A/T)A(A/T)(A/G)
Initiator – (C/T)(C/T)A+1N(T/A)(T/C)(T/C)

Required efficient initiation
Recognised by multiple factors (context dependent)

Question 9

Core transcription machinery

Answer 9

TFIID:

- TBP – binds to TATA box
- TAF – Regulates binding of TBP

TFIIB: binds to BRE box and positions RNA Pol2 at the initiator site
TFIIF: Stabilises interaction between TBP &TFIIB and attracts TFIIE and TFIIE: Attracts TFIIH
TFIIH: Unwinds DNA at transcription start point, phosphorylates RNA Pol II and releases it to initiate transcription.

Question 10

Mutations in TFII complex genes cause DNA repair disorders and premature ageing syndromes

Answer 10

Xeroderma pigmentosum (XP) – extreme sun sensitivity and multiple skin cancers

Cockayne’s syndrome (CS) – systemic disorders: premature ageing

Trichothiodystrophy (TTD) – brittle hair, nails, photosensitivity, intellectual impairment

Question 11

Tissue specificity – enhancers and insulators

Answer 11

Enhancers:
200-500bp
Contain multiple transcription factor binding sites
Distant from gene; require looping out of DNA
Can function in either direction

Insulators:
Regulate enhancers
Work through binding of insulator proteins
Can regulate multiple genes
Can act as chromatin boundary markers

Question 12

Chromosome territories, enhancers and insulators.

Answer 12

Enhancers recruit other proteins that cause DNA to loop, bringing mediator proteins in contact with transcription machinery.

Proteins that bind enhancers may be tissue specific.

Question 13

Transcription - elongation

Answer 13

Gross structural change in RNA Pol II marks the transition from an ‘initiating complex’ to an ‘elongating complex’

Polymerase creates an RNA copy of the DNA template

RNA pol II can pause when it meets secondary structure or regions with complex sequence context, so rates of transcription are not uniform.

Question 14

Steps in RNA processing

Answer 14

5’ capping – Addition of 5’ cap structure

Splicing – Removal of introns.

Polyadenylation – Addition of poly-A tail

Question 15

The 5’ Cap

Answer 15

Modified Guanine nucleotide with unusual 5’ to 5’ bond.

Regulation of nuclear export – nuclear export is regulated by the CBC (Cap Binding Complex) which is recognised by the nuclear pore for export.

Prevention of 5’ exonuclease degradation – looks like regular 3’ mRNA end to the degradation machinery.

Promotion of translation – by interaction with translation initiation factors

Promotion of 5’ intron excision – by interaction with spliceosome

Question 16

The pre-mRNA splicing reaction

Answer 16

A specific adenine within the intron attacks the 5’ splice site, cutting the backbone of the RNA
The cut 5’ end of the intron covalently links to the adenine, creating a loop
The released 3’-OH end of the exon reacts with the start of the next exon sequence, joining the two together
The intron is released as a lariat, and ultimately degraded

Question 17

snRNA and the spliceosome

Answer 17

Five small RNA molecules (< 200 ntds each) termed U1, U2, U4, U5 & U6 - small nuclear RNAs (snRNA)

Each snRNA is complexed with at least 7 protein subunits to form a small nuclear riboprotein (snRNP)

These snRNPs form the core of the spliceosome that performs the splicing reaction

Question 18

Splicing happens in the ‘speckles’

Answer 18

This figure shows the co-localisation of spliceosomal components and RNA in the nuclear speckles

Question 19

Alternative splicing is regulated by splicing regulatory proteins

Answer 19

Serine Arginine rich (SR) proteins.

Bind exon and intron splicing enhancer sequences
Enhance splicing
Also have roles in RNA stability and transport.

Heterogeneous ribonucleoprotein particles (hnRNPs)

Bind exon and intron silencer sequences
Inhibit splicing
Also have roles in RNA stability and transport.

Question 20

Exon splicing enhancers and silencers

Answer 20

ESE and ISE elements bind SR proteins to promote splicing processes.

ESS and ISS elements bind hnRNP proteins to inhibit splicing processes.

Splicing enhancer and silencer elements can be located far from splice sites but brought into proximity by looped 3D structure of RNA molecules.

Question 21

Polyadenylation

Answer 21

CPSF - Cleavage and polyadenylation specificity factor – identifies poly A signal
CFI, CFII – Cleavage factors – cut mRNA to allow addition of poly A tail
CstF – Cleavage stimulation factor – promotes cleavage by CFI and CFII
PAP - Polyadenylate polymerase – adds poly A
PABI and PABII - Poly A binding proteins – regulate mRNA stability

Question 22

RNA export and localisation

Answer 22

RNA is actively exported to the cytoplasm where it can be translated.

This is not a passive process; it involves RNA transport proteins (e.g. hnRNPC, hnRNPA, p15, TAP) that actively move the transcript through the nuclear pore.

RNAs can then be directed to specific cellular locations; usually directed by sequences in the 3’ untranslated region of the transcript.

This can be by directed transport using the cytoskeleton or by random diffusion and trapping on docking proteins.

Question 23

RNA processing is co-transcriptional

Answer 23

Transcription, mRNA splicing, polyadenylation all occur simultaneously.

Splicing, nuclear export, localisation and mRNA degradation are also linked.

NOT a simple pipeline.

Question 24

The genetic code

Answer 24

The genetic code is translated by transfer RNAs (tRNAs).

Each tRNA species is charged with an amino acid that reflects its anticodon (e.g. TTC = =Phe).

Question 25

Ribosomes are the translation machinery

Answer 25

Eukaryotic ribosomes are a complex of rRNAs and a total of 82 different proteins (ribonucleoproteins).

Composed of 2 subunits 40S and 60S (plus some accessory proteins).

Combine to form mature 80S ribosome ready for translational initiation.

Question 26

Translation

Answer 26

3 stages; initiation, elongation and termination.

Initiation
involves Initiation factors (eIF1, eIF4) and poly A binding protein (PABP), and the association of the ribosomal subunits.

Elongation
involves delivery of amino acids in the right sequence by charged tRNAs.
Peptide bonds then form between adjacent amino acids and the polypeptide chain begins.

Termination
occurs when a termination codon is encountered, which binds release factors (e.g. eRF1).
The final peptide bond is then hydrolysed and the polypeptide is released

Question 27

Sequences determining mRNA stability

Answer 27

RNA turnover can also be determined by RNA binding proteins (RBPs).

RBPs can bind specific sequences within the transcripts that regulate RNA stability.

AU-rich elements (AREs) bind RBPs to elicit degradation.

C-rich elements bind alphaCP proteins to stabilise mRNAs.

Question 28

Nonsense mediated decay (NMD)

Answer 28

An mRNA surveillance mechanism whereby transcripts encoding premature termination codons (PTCs) are selectively degraded.

PTCs can form by point mutations, frameshift events or splicing errors.

NMD occurs by virtue of interactions between the ribosome, exon junction complexes and degradation machinery.

Question 29

Small RNA regulation: miRNAs

Answer 29

20-22bp long

Each targets can target ~mRNA transcripts.

Can act in ‘modules’ – one mRNA targeted by several related miRNAs

Important in most cellular processes, but particularly development and cell cycle.

Question 30

Long non-coding RNAs : lncRNAs

Answer 30

Effects on Chromatin structure

Effects on transcription

Effects on Splicing

Effects on stability

Question 31

Conclusion

Answer 31

Regulation of gene expression is complex and multifaceted.

Genes can be regulated by:
Changes in chromatin architecture and epigenetics
Transcriptional changes
Changes in mRNA processing
Altered translation
Altered turnover dynamics
Small and long ncRNA regulation

These processes are interlinked so modification of one may affect others