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Pattys Lectures L18-22 Flashcards

1
Q

Is there proof-reading in transcription in Eukaryotes?

A

No

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2
Q

How many RNA pols are there in Euks Transcription?

A

3

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3
Q

What is the structure of RNAPs

A

2 large subunits and multiple (10-14) smaller subunits

Similar to proks

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4
Q

What is the CTD heptad repeat that distinguishes RNAPII from the other RNAPs?

A

YSPTSPS

young stinky prostitute tickles sams penile spasms

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5
Q

How many copies of the CTD are there in humans?

A

52
Can be a substrate for kinases
flexible scaffold dock for proteins
Proteins come on and off the scaffold via phosphorylation

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6
Q

How many NTs must RNAPII transcribe for it to escape?

A

5
4NTs= Escape commitment
>5NTs=Clash, Escape (8-9NTs)

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7
Q

After how many NTs does promoter-proximal pausing occur?
TFIIH phosphorylates CTD Ser5/Cap addition
NELF (negative elongation factor) and DSIF inhibit elongation

A

+20 to +40

Rate limiting step (check-point)
5’ CAP ADDITION

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8
Q

CTDK-1/P-TEFb

NB/ RNAPII is very processive

A

phosphorylates inhibitory proteins NELF/DSIF and ser2
NELF phosphorylation by P-TEFb releases elongation inhibition
DSIF phosphorylation by P-TEFb recruits elongation factors (elongin/ELL)
CTD phosphorylation by P-TEFb on Ser2 recruits factors for 3’ end formation

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9
Q

Pre-mRNA 5’ Cap addition

7-methyl guanosine m7G

A

Addition of m7G to 5’ end of pre-mRNA is known as capping
Capping is a co-transcriptional process
Capping enzyme complex is bound to CTD of RNAPII

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10
Q

What is the importance of the 5’ CAP?

A

Promotes mRNA stability by preventing 5’ end degradation
Promotes intron excision (mRNA splicing)
Acts as a tag to promote mRNA export from nucleus
Stimulates mRNA translation in the cytoplasm

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11
Q

3 step process: pre-mRNA 5’ Capping enzyme

A

bifunctional enzyme

1) NT phosphohydrolase
2) RNA guanylyl transferase

Guanine N7 methyl transferase
Nucleoside-2-O-methyl transferase

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12
Q

In Eukaryotes is there a transcriptional termination signal?

A

NO

3’ ends of mRNAs are formed by CLEAVAGE immediately followed by poly-adenylation

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13
Q

What is the importance of a polyA tail?

Poly(A) polymerase does not require a template

A

Promotes mRNA stability-protects from 3’ exoribonucleases
Promotes mRNA export
Affects translational EFFICIENCY (the longer the poly (A) tail the more efficient translation is)

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14
Q

How does termination occur in transcription? Eukaryotes

A

After the mRNA has been made the RNAPII continues beyond 3’ end cleavage and poly(A) tail addition. 3’ end cleavage allows entry of 5’-3’ exoribonucleases which act like a “torpedo” to degrade nascent RNA.
These exoribonucleases will eventually catch up with the RNAPII and will cause the complex to dissociate.

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15
Q

Do both Euks and Proks undergo splicing?

A

NOOOOOO
Only Euks
Pros are dependent on the genome they have

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16
Q

What are the main NTs in introns and exons?

A

introns (and promoters) -A/T rich

exons-G/C rich

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17
Q

Splice donor site and acceptor site?

Within intron

A

Donor-GU (100% invarient)
Acceptor-AG (99.9% conserved)
Branch point = unpaired A
Pyrimidine rich region adjacent to the A site will assist with the splicing reaction IN HIGHER EUKS with large introns

18
Q

Why is U5 snRNP important in lariat formation?

A

Once you have made the lariat you put the mRNA in a dangerous position to be degraded by exonuclease
U5 snRNP plays an important role and bridges exons that are going to be joined and keeps them in place in close proximity

19
Q

What is the debranching enzyme used for?

A

Loop/lariat is insensitive to exonucleases so need debranching enzyme to open up the lariat
It is now exposed and subjected to nucleases so we are able to reuse the NTs

20
Q

What type of disorders arise form cis or trans acting mutations?

A

Cis-loss of function/frame-shifts
Trans-Retinitis pigmentosa
Spinal muscular atrophy

21
Q

What proteins bind to ESEs?
Alternative splicing
exonic splicing enhancer

A 
SR proteins
serine/arginine
These bind to ESEs and recruits U2AF to upstream intron (recruits U2 snRNP)
Recruits U1 snRNP to downstream intron
SR proteins also promote mRNA export
22
Q

How is drosphila sex determined?

A

By a ration
X:A = # X chromosomes:sets of autosomes (A)
FEMALE (XX)=2:2 MALE (XY)=1:2
No dominant gene in drosophila whereas in humans dominant gene is on the Y chromosome

23
Q

What protein promotes female development in drosophila?

This protein has a premature stop codon in exon 3

A

Sxl (sex-lethal) - transacting factor
There are two promoters-early and late
Sxl early protein promotes alternative splicing of sxl late pre-mRNA
Sxl regulates its OWN pre-mRNA splicing
Sxl early is a SPLICING REPRESSOR -prevents U2AF from binding within intron2
Exon 3 out of 8 is skipped out in females by using sex early protein
Sxl early then produces functional Sxl LATE PROTEIN

24
Q

What other protein other than Sxl is needed for female development?

A

Tra
Gene only has 4 exons
Premature stop codon exists in exon2

25
What protein encodes 2 different function proteins with different splice variants and is the third pathway in the drosophila developmental pathway?
Dsx (Doublesex gene) Tra2 SR protein Exon skipping in males of exon 4 out of 6 exons Exon 4 has ESE site and Tra made previously in females is recruited to Tra2 SR protein present on Exon 4 ESE. This includes exon 4 Tra recruits U2AF to intron 3 Exon 4 uncovers a new poly(A) site Males exon1,2,3,5,6 Females exon 1,2,3,4Poly(A)tail
26
What is the cause of myotonic dystrophy?
``` Sequesters trans-acting splicing factors CUG repeats in the 3'UTR (non-coding seq) n=50-4000 n=38-49 premutation n=5-37 normal ``` 1. Expanded triplet repeat in 3' UTR forms HAIRPIN 2. Hairpin sequesters Muscleblind-like I splicing factor, which effects alternative splicing 3. Affects splicing of pre-mRNAs requiring these factors
27
Muscle blind like I splicing factor causes what diseases?
``` Insulin receptor:Insulin resistance Chloride channel:Myotonia Cardiac troponin T:Cardiac abnormalities Ryanodine receptor:Muscle weakness NMDA receptor 1:CNS effects ``` Myotonic dystrophy Muscle blind is involved in a lot of alternative splicing factors
28
B-RAF kinase B-RAF V600E What is it inhibited by?
Leads to inhibitor resistance BRAF involved in MAPK cascade RAS-BRAF-MEK-ERK-CCND1-cell proliferation p61 BRAF V600E alt splicing isoform lacks exons 4-8 and activates MEK- is oncogenic and becomes constitutively active RAS binding domain is deleted BRAF V600E is inhibited by vemurafenib Resistance to vemurafenib is associated with 61kDa variant of BRAF V600E arising from alternative splicing
29
RNA editing:mRNA variation not caused by alternative-splicing
Cytidine deaminase (occurs in the NUCLEUS) Changes NH2 to O In a deamination reaction it creates URACIL EXAMPLE:ApoB gene makes 2 different variants: Large-liver-fatty acid metabolism Small-intestine-dietry lipids, chylomicron proteins introduced immature stop codon CAA->UAA ADARs Adenosine deaminases acting on dsRNA Substrate is ds pre-mRNA (DOUBLE STRANDED) A changed to I (inosine) I is recognised as a G (CAG=CIG=CGG)
30
What are the consequences of adenosine demonises acting on dsRNA? ds regions caused by hairpins formed by ss pre-mRNA/mRNA
affect splice acceptor sites affect start/stop codons modify ion channel and neurotransmitter function defence against viruses (change of seq will change immune system,-protection of viruses)
31
mRNA export into cytoplasm
Through nucleopores since mRNA is too large to diffuse from nucleus to cytoplasm mRNA goes head first (5' CAP)
32
mRNA export by heterodimeric transport receptor
Nxf1/TAP and Nxt1 heterodimeric subunits of mRNA export receptor complex SR proteins are dephosphorylated after splicing-permits binding of exporter complex REF (RNA Export Factor) protein coordinates mRNA splicing and export Marks EJC (exon-exon junction complex) at completion of splicing Bridges mature mRNA and Nxf1/TAP exporter REF protein is also recognised by the transporter complex
33
In mRNA export how is the CAP protected in the cytoplasm?
CAP gets protected by binding a translational complex called Euk initiation factor 4E EIF4E In the cytoplasm mRNA undergoes an exchange of proteins PABPII to PABPI
34
Protecting mRNAs from degradation
``` 5' M7G CAP 3' Poly(A) addition PABP bound to poly(A) tail (mRNA circularisation in cytoplasm) Protects against 3'-5' deadenylases NUCLEUS-PABPII CYTOPLASM-PABPI ```
35
Cytoplasmic mRNA decay The degradation reactions will take place in specific compartments in the cell (both nuclear and cytoplasmic compartments are involved)
Poly(A) tail has protective function Poly(A) Nuclease (PAN) will deadenylate the mRNA (remove the tail) - PABPI comes off De-capping protein DCP1 removes the CAP Allows exoribonucleases to chew up the rest of the mRNA 5'-3' exo = XRN1 or GW/P-body 3'-5' exo = Exosome
36
What do PREs and AREs do?
PRE-pyrimidine rich element inhibit deadenylation ARE-AU rich element enhance deadenylation
37
Name 2 examples of mRNA diseases?
C-fos (proto-oncogene) normally short lived t1/2=1hr 2AREs de-stabilise mRNA Deletion of ARE stabilises mRNA Upregulation of mRNA-malignant skin cancer Beta-globin normally long lived t1/2=20-30hr Reduction causes thalassemia Disrupt PRE in beta-globin Reduce mRNA stability
38
Cytoplasmic polyadenylation involves what proteins?
Cis-acting CPE (cytoplasmic polyadenylation element) in the 3' UTR affects polyadenylation Affected transcripts are usually products of spermatogenesis and oogenesis in which poly(A) tails are removed after nuclear export These mRNAs are stored in a translationally dormant state Cytoplasmic poly(A)+ addition makes stored mRNA competent for translation-process is faster than de novo transcription
39
What are the 3 types of mRNA quality control?
- Non stop mediated decay - Nonsense mediated decay (NMD) - No-go decay Depends on the type of error in the mRNA Processes usually involved in the pioneering round of translation (FIRST round)
40
Why are histones an exception?
Get their poly(A) tail added in the cytoplasm whereas most mRNA get their tail added in the nucleus
41
Gene expression and mRNA localisation - what mRNAs are recruited to the - and + end of the motors? Dosophila
- end dynein motor, BICOID driven to anterior end + end kinesin, OSKAR driven to posterior end Actin, needed at leading end of fibroblast and it is localised to mRNA at that edge (leading) so you can make a protein where it is needed Cis acting sorting signals (zipcodes) (usually in 3' UTR) recruits trans-acting factors, which promote transport of the mRNAs along MTs or actin to the place they are needed

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