Lecture 25 Flashcards
(14 cards)
Alternative translation start sites
Some mRNAs lack 5’ cap so, there’s alternative initiation sites called internal ribosome entry site (IREs) which are on 5’ side of the start codon. Functions similar to 5’ cap as it can bind to IFs and small ribosomal subunit (eiF4F and 40S small subunit). Everything afterwards is same.
Experimental application of IREs
IREs allow the creation of polygenetic mRNA in eukaryotes. You can fuse a reporter protein to gene of interest or insert IREs after gene of interest and put reporter protein gene after which may stop poly(A) tail insertion. When transcripted you have polygenetic mRNA made. mRNA of interest is translated but reporter mRNA is translated through CAP-independent mechanism via IREs to make promoter protein. Can track target protein.
Disadvantage of reporter gene
disadvantage is that big bulky group can impact native structure/function of protein and reporter may not function as well when bound.
Non-stop mRNA decay
Detects/destroys mRNA that lacks stop codon. W/o s.c ribosome begins and continues translation until 3’ poly(A) tail is reached. Poly(A) codes for lysine. With PP, there will be a series of lysine at 3’ end. Ribosome stops at end of mRNA stalling it and signalling to the cell the mRNA is defective. A protein binds and recruits more protein that triggers the dissociation of ribosomal complex from mRNA and degrades mRNA, breaking as 3’-5’ exonucleases. Defective protein recognised by C-terminal polylysine tag and proteases degrades it.
Nonsense-mediated mRNA decay
Mutations leading to premature stop codon in mRNA leading to truncated protein therefore, very bad. Under normal circumstances, introns are spliced and exons are ligated by spliceosome. In doing so, spliceosome marks boundaries between exons. Markers known as exon junction complex. First translation of mRNA, initiation occurs and as it translates EJC removed. At the end there won’t be any EJCs left. A defective nonsense mRNA will result in residual EJCs as they haven’t been translated. EJCs signal to cell machinery to recruit additional proteins that bind and recruits decapping enzymes. They remove 5’ cap to allow the mRNA to degrade by 5’-3’ exonucleases. Takes it out of circulation.
No-go mRNA decay
Ribosome stalls before SC reached due to:
- Presence of rare codons: not the preferred codon for a protein therefore, no tRNA for codon.
- Secondary structure formation: Internal base pairing in mRNA form stem loop structure, adding bulk. Once ribosome complex reaches it, it stalls.
It recruits factors that recruit other proteins to dissociate ribosome from mRNA. Enzymes cleave mRNA and chew it up.
Small RNAs regulate mRNA stability
We want to control mRNA half life to control protein levels. Called RNA interference. Triggered by double-strand RNA (dsRNA) which comes from exogenous source (virus of experimental or endogenously generated by RNA Pol. 2 and 3). RNA polymerase 1 can protect from retroviral mRNA.
Exogenous dsRNA
Cleaved by RNAse H called Dicer into 21 nt-long fragments. One of the single strands is left as cleavage product.
SiRNA
Loaded to argonaute proteins forming RISC
RISC
Via RNA, RISC can recognise specific RNA products in cell, bind via complementary sequences and stop protein transcription by being bulky or starts cleaving mRNA.
miRNA
Micro RNAs are generated from larger transcripts made by RNA pol. II or RNA pol. III. Transcript made where base pairings form short hairpin loop. This causes DICER to cleave it and form double-stranded RNA. dsRNAs processed into single strand RNA, RISC formed and RISC binds to target mRNA, inhibiting protein synthesis.
Increase iron
Increase iron = decrease transferrin receptors = increase ferritin.
Ferritin synthesis
Ferritin mRNA has stem-loop called the iron-response element (IRE) in the 5’ untranslated region which binds to IRE binding protein (responsive to Fe levels). IRE upstream of coding region. When [Fe] low, IRE binding protein binds to ferritin mRNA and blocks translation initiation. When [Fe] high, IRE binding protein has increased affinity for Fe than the mRNA therefore, binds to Fe. Once bound, conformational changes prevents it from binding to IRE. Translation occurs producing ferritin.
Transferrin receptor synthesis
The transferrin receptor mRNA has several IREs in its 3’ untranslated region. When iron levels are low, IRE-binding proteins attach to these IREs. Because these IREs are at the 3’ end, they do not block translation, but instead protect the mRNA from being degraded, increasing its half-life and allowing more transferrin receptor to be made. When iron levels are high, iron binds to the IRE-binding proteins, preventing them from binding to the IREs. Without this protection, enzymes can degrade the mRNA, so less transferrin receptor is produced.
