Lecture 3 - Gene Expression and Regulation Flashcards
Chromatin architecture and gene expression
Chromatin structure must be unwound to allow transcription factors and other regulatory proteins access to DNA.
How is chromatin structure controlled?
Histone modifications
DNA methylation
Histone modifications
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).
DNA methylation
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.
Nuclear organisation and chromosome territories
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.
RNA is transcribed by RNA polymerases
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.
Transcription Initiation
Transcription is regulated by specific proteins (transcription factors) binding to specific sequences (promoters or enhancers).
Core promoter sequences
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)
Core transcription machinery
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.
Mutations in TFII complex genes cause DNA repair disorders and premature ageing syndromes
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
Tissue specificity – enhancers and insulators
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
Chromosome territories, enhancers and insulators.
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.
Transcription - elongation
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.
Steps in RNA processing
5’ capping – Addition of 5’ cap structure
Splicing – Removal of introns.
Polyadenylation – Addition of poly-A tail
The 5’ Cap
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
The pre-mRNA splicing reaction
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
snRNA and the spliceosome
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
Splicing happens in the ‘speckles’
This figure shows the co-localisation of spliceosomal components and RNA in the nuclear speckles
Alternative splicing is regulated by splicing regulatory proteins
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.
Exon splicing enhancers and silencers
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.
Polyadenylation
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
RNA export and localisation
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.
RNA processing is co-transcriptional
Transcription, mRNA splicing, polyadenylation all occur simultaneously.
Splicing, nuclear export, localisation and mRNA degradation are also linked.
NOT a simple pipeline.
The genetic code
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).
Ribosomes are the translation machinery
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.
Translation
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
Sequences determining mRNA stability
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.
Nonsense mediated decay (NMD)
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.
Small RNA regulation: miRNAs
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.
Long non-coding RNAs : lncRNAs
Effects on Chromatin structure
Effects on transcription
Effects on Splicing
Effects on stability
Conclusion
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
