BCMB30012 Notes
4
M ODULE 1: G ENE R EGULATION
- Eukaryotic gene expression at every level:
o Chromatic structure à histone modification, methylation of CpG, lncRNA (long-non-coding RNA) o Transcription à initiation and elongation regulation
o mRNA processing à splicing
o mRNA stability à miRNA, RNA binding proteins o Translation
o Post-translational modifications à phosphorylation, acetylation, ubiquitination, etc.
o Protein transport o Protein degradation - Transcriptional regulation:
o Eukaryotes have 2 types of positive regulation:
§ Molecular signal causes disassociation of activator from DNA à inhibits transcription
§ Molecular signal causes binding of activator to DNA à induces transcription
Bacterial gene expression regulation Eukaryotic gene expression regulation Minimal chromatic regulation Lots of chromatin regulation
Normal gene expression high à must be downregulated Normal gene expression low à must be upregulated Small promoters and operators Large promoter regions (basal promoter + large gene
regulatory region)
Lots of repressors Lots of activators
Operons à multiple genes regulated together Each gene has its own promoter
Transcription and translation regulated together Transcription and translation regulated separately Small non-coding RNAs and RNA binding proteins miRNAs and RNA binding protein
No splicing Splicing
- Translational regulation:
o Eukaryotic mRNA forms a ring structure:
§ UTR = Un-Translated Region
§ Polysomes à multiple ribosomes are bound to the mRNA at once à quick translation of lots of protein
§ Proteins can bind to the UTR regions and regulate translation à Proteins bound to 3’UTR can repress or activate translation because they are close to the 5’ end
o Changes in mRNA stability:
§ Stability of mRNA depends on PolyA tail à longer tail, longer half lives
§ At ~25 As the mRNA starts degrading more rapidly
o Regulation of Iron intake:
§ Iron is essential and toxic à can cause oxidative stress and damage to the cells if allowed to travel the blood freely or build up in tissues
BCMB30012 Notes
5
§ Fe3+ is bound to Transferrin à Transferrin binds to the Transferrin Receptor which is endocytosed into the cell alongside the Fe à Fe3+ is stored in Ferritin and released into the cytosol (for Fe dependent enzymes)
§ Regulation of Ferritin
• Low iron à don’t need as much Ferritin for storage à Iron Regulatory Protein (IRP) binds to Iron Response Element sequence in 5’UTR and stops translation
• High iron à need more Ferritin to store the iron à IRP is inactivated by Fe3+ and does not bind IRE à translation proceeds
§ Regulation of Transferrin Receptor
• Low iron à the cell needs to intake more iron à need more receptor à IRP binds to IRE in the 3’UTR and stabilises the mRNA à translation proceeds
• High iron à cell doesn’t need to intake more iron à IRP is inactive by binding to Fe3+ and doesn’t bind the IRE at the 3’UTR à nothing is keeping the mRNA stable à mRNA degraded by endonucleases and/or miRNAs
- RNA editing after transcription:
o tRNAs and rRNAs are usually edited and folded for their functionality o mRNA sequences can also be edited:
§ ADAR à Adenosine Deaminases Acting on RNA à deamination of Adenosine (A) to Inosine (I) à e.g. ion channel in brain
• Changes the coding à A binds only with 1 other base but I has the option of 3
• Can create a STOP codon and truncate the protein
• Alter splicing
• Cause binding patterns e.g. hairpin loop
§ Cytidine to Uridine editing à e.g. apolipoprotein mRNA (introduces STOP codon)
o Possible roles à protection from retroviruses which can inject their RNA into a cell after infection or forming alternative proteins from a single RNA, not really sure why
- Techniques to examine gene expression:
o If you’re trying to examine mRNA à RT-PCR (1 gene) or RNA-seq (looks at entire sequence/transcriptome)
o If you’re trying to examine protein à WB (1 protein) or Proteomics (looks at all proteins in a sample/proteome)
- RT-PCR:
o 1. Extract RNA from sample o 2. Treat with DNase I
o 3. Use spectrophotometer to test conc. and purity o 4. Run RT-PCR
§ Use oligo-dT primer to anneal to mRNA poly-A-tail
§ RT + dNTPs create hybrid DNA:mRNA
§ mRNA is degraded with alkali
§ DNA pol I + dNTPs + primers create the cDNA
