What post transcirptional processing does tRNA go through?
Methylation
Psuedourdylation
- isomer of nucleotide uridine=psudouridine
- post transcriptional isomerisation of specific uridine molecules.
- most prevelent of over 100 different nucleotides found in RNA
What post transcriptional processing does rRNA go through?
Cleavage
methylation
Where are eukaryotic ribosomes made and why is no translational amplification needed?
nucleolus - site of rRNA processing and assembly into ribosome units. (nucleolus is not membrane bound)
200 tandem repeats of 455 rDNA in H.sapiens.
2000 copies of 5S rDNA transcribed by RNAP111
No transcriptional amplification is needed.
How is ribosomal rRNA processed?
-following transcription of pre rRNA from rDNA in nucelolus the pre rRNA is modified and associates with some ribosomal proteins
- small nuclear RNAs (snoRNA dictate the modifcications by base pairing with target sites in eukaryotic pre rRNA and may also play a role in folding and methylation.
- cleavage at various sites gives rise to 18S and 58S and 28S
- last steps of rRNA processing require changes to generate mature 5.8S and 38S rRNA
-Form SSU and LSU via protein addition and spontaneous assembly
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How does the anatomy of mRNA differ in bacteria and eukaryotes?
Bacteria contain a shine delgardo sequence which helps point out the start codon.
Bacteria mRNA often polycistronic - containing several coding regions
Eukaryotic 5’ UTR contains a M^7G cap
Eukaryotic 3’ UTR contain a 3’ UTR Poly A tail
In what domain of organisms does mRNA processing occur in?
Eukaryotes
- both eneds are modified
- capping on 5’ end.
- polyadenylation on 3’ end.
- allows cells to assess whether both ends present before export from cell.
Why is the 5’ cap important in mRNA?
- signifies end of eukaryotic mRNA and helps to distinguish mRNA from other types of RNA which do not have a cap, eg. RNAP1 and RNAP111 are uncapped.
- it also binds a protein complex called the CBC - cap binding complex in the nucleus which helps RNA to be properly processed and exported.
- Also has an important role in translation in cytosol.
How is the mRNA cap formed?
- after RNAP11 has produced about 25 RNA nucleotides 5’ is modified
- cap consists of modifed guanine nucleotide
- 3 enzymes perform capping reaction
*phosphatase removes phosphate from 5’end RNA
*Gyanyl transferase adds a GMP in 5’->5’ reverse linkage. - methyl transferase adds a methyl group to guanosine.
= 7-methylguanosine in the 5’-5’ linkage.
What are the 3 enzymes used in cap formation?
Phosphatase
Guanyl transferase
Methyl Transferase
What is the poly A tail important for?
- slows degradation in the cytoplams
- mRNA’s wont go through nuclear pores without PA tail.
What happens to RNA after it emerges from RNAP11?
- CStF - cleavage stimulation factor and CPSF - cleavage adn polyadenylation specificity factor are transfered to 3’ end of processing sequence as they recognises sequences.
- these bind to specific nucleotide sequences on the emerging RNA molecule and additional proteins assemble with them to create the 3’ end of the mRNA
- first RNA is cleaved- terminating transcription, endonuclease cleaves.
- poly A polymerase then adds one at a time approx 200A nucelotides to 3’ end produced by cleavage.
- nucelotide precursor is ATP
- Poly A tail does not encode template so is not encoded in the genome.
- As PA tails synthesized some Poly A binding proteins PABPs remain bound to tail- forming a ribonucleoprotein.
How is the poly A tail removed in the cytoplasm?
DAN burns away PA tail in cytoplasm so mRNA can be destroyed by exosome.
What does the length of the poly A tail determine?
mRNAs stability in eukaryotes.
Why does the histone mRNA lack a poly A tail? What does it have instead?
Has a much less stable stem and loop.
as during synthesis stage the cell needs to make alot of histones, however as soon as replication stops protein needs to stop making histones to prevent wase.
- Therefore the protein that helps stabilise and bind mRNA gets phosphorylated at the end of DNA replication and degrades mRNA.
What do they have in additon of DAN in RBC cytoplasm?
PAP - which increases the length of poly a tail
tug of war between dan and pap
What do sequences in the 3’ UTR of mRNA do?
Give an example
target mRNA to specific places in the cytoplasm
eg. nerve cells are massive.
- wouldnt make sense to translate in cell body if needed in axon
- more sense to make mRNA and translate in axon where needed.
- this requires polyA and motor proteins.
- So PABP binds to cytoskeletal motors.
Vasopressin ( an antidiuretic hormone ADH) mRNA is directed to dendrites in they hypothalamus.
How is iron uptake regulated in the blood?
- Transferrin takes iron up from the blood
- Ferritin stores and releases iron in a controlled fashion.
- cytosolic aconitase doubles up as iron regulatory protein.
- During iron depletion
- cytosolic aconitase binds to ferretin mRNA at RBS which blocks ribosome from binding and binds transferrin mRNA which blocks endonuclease site and increases half life.
- when level increase the protein dissociates from both mRNA’s leading to degradation of transferrin mRNA and expression of ferritin mRNA
Why would excess iron be a bad thing?
it catalyses the formation of reactive oxygen species that cause damage.
What do iron regulatory proteins do? IRP
regulate iron concentrations and post transcriptionally regulate expression of iron metabolism genes in order to optimise iron availability
- control iron metals by binding to specific noncoding sequences in mRNA called iron responsive elements IREs
which occur in either the 3’UTR or 5’UTR or mRNA
eg. ferritin adn transferrin contains IRES
Give 2 examples of RNAi’s which are able to regulate other RNA’s by binding to them and inhibit gene expression? How do they typically do this?
microRNA miRNA
small interfereing RNA siRNA
^ both are central to RNA interferance
they can both bind to mRNA and increase or decrease their activity. although they typically act by causing destruction of specific mRNA molecules.
How many of human protein coding genes does miRNA appear to regulate?
1/3 of all
How are miRNA precursors synthesized?
by RNAP11 and capped and polyadenylated.
they then undergo special processing after which the miRNA is assembled with a set of proteins to form a RNA induced silencing complex. or RISC
What does the RISC do once formed?
- seeks out target RNA’s by searching for complimentary nucleotide sequences.
- search facilitated by Argonaute protein - component of RISC which displays the 5’ region of miRNA so that it is optimally positioned for base pairing.
- ussually bp takes place in 3’ utr of target mRNA
What are the 2 outcomes once miRNA is bound to mRNA?
- if the base pairing is extensive then the mRNA is cleaved by the Argonaute protein, removing the Poly Adenosine tail and exposing mRNA to exonucleases for degradation.
- following cleavage of mRNA RISC with its associated miRNA is released to seek out additional mRNAs. - if bp is less extensive argonaute does not slice the mrna but tranlsation of it is repressed and mrna is destabilised.
