BIOL253 L15

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  • Created by: Katherine
  • Created on: 21-04-17 15:27
What is cleavage?
When the strand of DNA is cute
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What is splicing?
Where part of the DNA is removed (E.g. the intron)
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What is 5'capping?
Where the sequence is capped
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What is polyadenylation?
Where Adenine groups are added
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What are the 3 types of RNA editing?
Base insertion, base deletion and base modification
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What is base insertion?
WHere a base is inserted
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What is base deletion?
Where a base is deleted
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What is base modification?
Where a base is substituted (modified/swapped for another base)
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Capping is unique to...
Eukaryotes
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What are the functions of the 5' cap?
Protection of mRNA from degradation, translatability - presence of the cap stimulates translation of mRNA (300 fold), transport from the nucleus to the cytoplasme
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The presence of a cap structure....
Improves translatability
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mRNAs which are not capped are...
Not translated very effectively at all
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Why does the cap need to be present?
For the RNA to be transported
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Capping involves how many steps?
3
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What are the steps of capping?
An RNA triphosphatase removes the terminal phosphate at the 5' end. Guanylyl transferase used GTP to attach GMP. The guanine is methylated by a methyltransferase.
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Once rna polymerase gets to the end of the transcript a partiular sequence is recognised. Which part is the sequence for cleavage?
CA site
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What does the cleavage mean?
It means that the bit needed is clipped off
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What happens after cleavage?
A complex comes together
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What is the enzyme which adds multiple adenosines in a row?
Poly A polymerase
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Most RNA in our bodies require...
Splicing
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The more complicated the organism, the more introns which
Need to be removed (spliced)
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What are introns?
Parts of the DNA which were encoded in the genome but the cell doesn't want to be synthesised
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What are the reactions that remove introns called?
Transesterfification reactions - most are removed by the spliceosome, although some are self splicing
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What is the basic mechanism of splicing
Within our intron sequence, we have a particular adenzine - you get nucleophilic attack between the hydroxyl and the 5' end of the intron. You then get a further nucleophilic attack from the 3' hydroxyl of exon 1 to the 5' end of exon 2. You join th
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What happens whne the 3' end of exon 1 has a nucleophilic attack towards 5' end of exon 2?
They join togeth and the intron is excised as a lariat structure followig two consecutibe transesterification reactions
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What are introns defined by?
The presence of a 5' GU and a 3' AG each of which is found within a particular sequence context
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The splice site boundaries are recognised by...
the spliceosome
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What is the spliceosome composed of?
5 small nuclear ribonucleoproteins (snRNPs) each of which contains a snRNA and proteins
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What are the key bits to take away?
It is catalysed by the spliseosome, the spliceosome consists of proteins and small nuclear RNAs which serve to bring the ends together and create a structure that facilitates the two sesquential transesterification reactions
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YOU DON'T NEED TO MEMORISE THE MECHANISM WITH ALL THE Us
YAY
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What is the EJC complex?
The exon junction complex - it is left at splic junctions after splicing
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What does the EJC do?
It marks the transcript as processes and interacts with export and translation proteins.
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Why is the EJC important?
It prevents incompletely processed RNA from being exported
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Splicing together exons in different ways means that...
you can produce loads of different compounds
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By using alternative splicing, you can have
DIFFERENT VERSIONS OF THE MRNA AND UTIMATELY DIFFERENT PROTEIN
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What does regulation of alternative splicing result in?
Distinct mRNAs frrom same pre-mRNA, developmentally controlled splicing, organ/tissue specific splicing
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What is key to the co-ordination of all of the processes? To make sure that they all occur?
The c terminal domain of the RPB1 component of RNA polymerase
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The capplic complex is recruited by...
the patrially phosphorylated CTD and it tells it that transcrption has started.
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The CTD recruits the capping enzyme complex and you get..
Additional phosphorylation of the CTD upon transcription elongation which allows recruitment of splicing machinery
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What is RNA editing?
A process that adds/deletes bases from pre-mRNA or chemically alters bases, resulting in an mRNA with bases that don't match its DNA sequence
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What are trypanosomes?
Unicellular parasites
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What do trypanosomes cause?
African sleeping sickness (from tsetse flies)
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How many mitochondira do trypanosomes have?
1
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What is interesting about trypanosome RNA?
It undergoes substantial editing before it can be used
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How does RNA editing occur?
As well as transcribing mRNA, it also transcribes gRNA. gRNA have regions of complementarily to mRNA and themselves
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What happens to the mismatch?
Endonuclease cuts mRNA at mismatch and it allows uridyly transferase to dd Us to the 3' end of the 5' mRNA fragment, guided by the gRNA.
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What are the key points of RNA editing when it needs to insert nucleotides?
Guide RNA aligns and indicates the site where cleavage has to occur. An then, within this bit which was previously looped out you have a templte which serves to tell uridylyl transferase it needs to insert some Us. THis leaves a nick, ligase joins
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What are the key points of RNA editing when it needs to remove nucleotides?
If one of the bases has been looped out, it is recognised by an endonuclease which cuts the mrna, and recruits a exonuclease whcih removes it out of the mrna - and then it's all joined together by ligase
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What type of RNA editing is done by humans?
Substitution editing
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Why is RNA degradation important?
It controls how much the transcripts can be used to produce proteins, so it's another level at which gene expression can be controlled.
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Do RNAs all have the same stability?
No - the half lives can vary
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Give a determinant of stability?
The presence of the poly-A-tail.
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How does the presence of the poly A tail determine stability?
Tail removal is directed by AU rich elements. The AU rich elements provide a signal to the cell, saying to remove the adenosines from the mRNA - it messages that this is a mRNA that the cell only needs for a little while
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How is cfos deregulated?
cfos gene is picked up by a virus, and a consequence, the are sequence is lost, so the virus encodes a varient of fos that has a long half life.
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What happens if RNA doesn't have an ARE?
It has prolonged survival and more protein is produced
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What is a particular targeted form of mRNA degradation?
RNAi (RNA interference)
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How was RNA i first identified?
In C.elegans. It was injected into the worms - found the corresponding gene was downregulated.
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How many types of RNAi are there in eukaryotes?
2
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What are the types of RNAi in eukaryotes?
siRNAs and miRNAs
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What do RNAis do?
The interference is able to destroy mRNA
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What happens in RNAi?
The double stranded RNA is cleaved by dicer (RNase) to make short double stranded RNA molecules. These are loaded onto a protein caused argonaute, where one strand is degraded to form a RISC complex. The complex then lines up on the RNA, degrades it
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What are miRNAs produced by?
RNA polymerase II so have a 5' cap structure and a poly A tail. They form a hair pin.
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The 5'3' end have been clipped off by a complexcalled
Drosha
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THis produces:
Precursor RNA
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What happens to the precursor RNA
It is transported from the nucleus to another complex 'dicer'
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What does dicer do?
Clips of the rest of the hairpin, leaving behind a duplex.
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What else is a substrate for dicer?
siRNA
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What is the difference between miNA and siRNA?
miRNA are imperfectly base paired while siRNA are perfectly base paired
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Card 2

Front

What is splicing?

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Where part of the DNA is removed (E.g. the intron)

Card 3

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What is 5'capping?

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Preview of the front of card 3

Card 4

Front

What is polyadenylation?

Back

Preview of the front of card 4

Card 5

Front

What are the 3 types of RNA editing?

Back

Preview of the front of card 5
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