RNA biology lecture 8 Flashcards
1
Q
Eukaryotic vs prokaryotic translation initiation
A
- Organisation of genes on mRNA = different
- prokaryotes = mRNA poly-cistronic, each gene has SD, co-transcriptional translation
- Eukaryotes = mRNA monocistronic, 1 gene but through alternative processing make ↑ mRNA, 5’ cap + 3’ tail
2
Q
Translation initiation in prokaryotes
A
- SD/rbs = us of Aug start
- 16S RNA associates via compl. RNA-RNA interactions
- This also positions P site of ribosome in region of AUG
- Additional factor join
- Complex recognised by 50S, finalise assoc. of ribosome onto ORF of mRNA
- Release 1F1/3 to open up A site
- ↑ ORF can be translated from 1 mRNA
3
Q
Translation initiation in eukaryotes
A
- 5’ cap + 3’ tail for regulation efficiency Cap-5’UTR-ORF-3’UTR-tail)
- CAP binds w/ CBP20/80, poly tail = occupied by PABP
- CBP + PABP interact w/ each other → circular mRNA
- In yeast, mRNA exported circular
- In cytoplasm, CBP + PABP exchanged for cytoplasmic versions
- Initiation = dependent on factors, eIF4F = 3 components, make up CAP-binding complex (cyt)
- eIF4e contacts CAP, eIF4G enables bridging btw PABP + eIF4E
- Mechanism = disc of 50S ribosome → 60S + 40S subunit → 40S trapped, 40S-3-IA assoc w/ ternary complex → 43S complex, mRNA occupied by 4F CAP binding complex, this complex assoc w. 43S → 48S ribosomal subunit, 48S scans RNA for AUG, at AUG forms complex where 60S joints → 80S initiation complex
- In eukaryotes, assoc of ribosome + cap means Aug can be 1000s of bp away from CAP site
4
Q
Translational control
1. Global
A
- E.g. by phosph of translational apparatus like initiator factors
- Formation of 3o complex, EIF2-GDP needs GTP, phosph of a-subunit of eIF2 on Ser51 → inhibition as eIF2B-catalysed exchange of GDP for GTP x
- Kinase = way regulation = integrated
- E.g. oxidative stress activates HRI, respond to environmental changes
- When goes wrong: x phosph Ser51 of eIF2 → T2D, sub of Ser51→Ala in mic → glucose intolerance
E.g. association of CAP
- Unphosph eI4F4e binds some CAPs, others only bind phosph CAP
- eI4F4E = phosph on Ser209
- 4E-Bp associates w/ eIF4E which blocks interaction btw eIF4e + eIF4G, preventing formation of eIF4F + recruitment of 43S
- phosph of 4eBP by mTOR Δ structure of 4E-BP → releases eIF4fe, associates w/ eIF4G
- Insulin signalling → MAPK → MTOR phosph → 4E-BP phosph, CAP x form
5
Q
Translational control
2. Individual
A
- Circular structure of mRNA = key as brings sequence in 3’UTR close to cap
- Regulation of mRNA w/ TOP sequence (found in proteins assoc. w/ translational apparatus, TOP mRNAA have C followed by 4-14 pyrimidine, unusual 5’, in growing cells, translated w/ ↑ efficiency)
- Regulation at transcript level = assoc w/ regulation of intracellular [ion]
- Ferritin + ferritin encoded protein sequester ion
- IRP = if iron around associates w/ it, if x IRP=free
- Iron dangerous, produces free radical
- Ferritin encoded by mRNA w/ classic stem loop, has bs for IRP (only binds when x bound IRP)
- Hinders assoc w/ 5’ UTR of ferritin, blocks translation initiation
6
Q
Translational control
2. iii Control of factors by binding UTR
A
- Sequence elements that can be recognised by SXL
- Dosage compensation in Drosphilia ↑ transcriptional output from genes from signal X chromosome in males= 2X in female
- Dosage compensation in females = prevented by repression of MSL2 by SXL
- SXL binds 3’UTR + recruits protein that prevents assoc of 43S w/ 5’ cap, also binds to 5’UTR
7
Q
Translational control
2. iv Role of CPE in translation inhibition
A
- Some maternal mRNAs not only involve controlled adenylation of mRNA, also CPEB-mediated inhibition of translation initiation
- Fertilisation = development driven by maternal mRNA, period of transcriptional inactivity
- In oocyte cytoplasm, mRNA transcripts have 3’UTR elements like CPE that sequesters CPEB, inhibits assembly of complex that makes functional CAO
- When signals activate kinases, phosph CPEB → inactivation of pARN, activation of Pol that extends poly A tail
8
Q
Translational control
2. v Translational regulation by MiRNA
A
- miRNA associate w/ complex in cytoplasm that enables them to associate w. compl sequences mostly in 3’ UTR
- Can inhibit Cap recognition or repression 60S joining
- Can repress translation at post-initiation stage by slowing elongation
9
Q
Translational control
2. vi Selenocysteine
A
- 25% proteins incorporate selene-cysteine at UGA stop
- Controlled by SECIS structure in 3’UTR that binds SEBP2 + tRNAsec allows incorp into peptide at stop
- 3’ UTR sequences can make specific proteins
10
Q
RNA degradation
A
- CAP + polyA tail protect proteins, need to remove
- PARN - 3’-5’ exoribonuclease degrades polyA tail, free 3’ targeted by EXOSOM
- Decapping E removes guanosine cap, exposes free 5’ end, degraded by 5’-3’ exoribonuc like XRN1
- Deadenylation independent pathways, mRNA cut in 1/2 w/ endonucl, exposes unprotected 5’ on 31/2 targeted
11
Q
Regulating stability of mRNA
A
- Key for overall expression levels
- mRNA stab + de-stab cis elements that control expression, if ↑ stable, repeatedly translated
- e.g. destabilise sequences like ARES = in 3’UTR, interact w/ proteins that recruit components of decay machinery, stability = Δ by factors that compete for ARE biding
12
Q
Nonsense mediated decay
A
- Assoc w. degradation of aberrant mRNA e.g. premature stop
- EJC denotes exon-exon junction
- After 1st round of translation, mRNA engages w. cytoplasmic CBP + PABP → repeated rounds of translation
- Immature stop codon likely flanked by EJC
- In primary round, stop is recognised as stop codon, release factors assoc w. ribosome
- Us of EJC, cells signal faulty mRNA transcript, recruit endonuc + upf1
13
Q
mRNA as regulatory target
Iron metabolism
A
- Translation initiation inhib by IRP
- IRP also recog 3’UTR of Tfr
- Intracellular Fe brought into cell by serum transferring
- Transferrin receptor assoc w/ iron → release Fe in cell
- Tfr mRNA has stem loop in 3’UTR, prevents mRNA degraded by exonuc
- ↓ Iron, need ↑ Tfr to get more iron in, IRP free, assoc w/ 3’ UTR of Tfr, prevents endoncucl
