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Flashcards in Lecture 14 Deck (18):

Core RNAP complex and GTFs:

- Recognises upstream control sequences (UCS)
- Upstream binding factor (UBF) recognises UCS


Ribosomal RNA genes:

- Repeated clusters of rDNA repeat unit (can be hundreds of copies!)
- Each is transcribed into a pre-rRNA precursor which is process into 18S, 5.8S and 28S rRNAs
- Transcription occurs in the nucleous (this is where ribosomes start, but they translate in the cytoplasm)



- Has TFIIIA, B an C, and also has a TBP component (which has evolved)
- Involved in processing Small RNAs include 5S RNA, snNAs, snoRNAs, gRNAs



- TBP recognises TATA box in the binding complex:
1. Complex recruits TFIIA and B to the promoter
Recruit RNAP
2. TFIIH and TFIIE melt DNA
3. Transcription initiates
4. RNAP tail is modified, important for processing steos


The primary transcript is processed in the nucleus:

- Capping
- Splicing
- Polyadenylation and termination


C terminal domain of RNAP consists of a series of 7aa's. This makes up the tail. Unphosophrylated:

- RNAP is in the pre-initiation stage, and doesn't recruit processing factors
- When RNAP reaches serine 5 it is phosphorylated and recruits the capping enzymes
- Dephosphorylation of this serine 2
- C terminal repeat will recruit splisosomal machinery and as RNA is made the introns are chopped ot.


Capping process:

- 5' end comprised of 7 methyl guanine and a triphosphate bond to a 5' hydroxyl group of mRNAs
- This is driven by other enzymes (not polymerases)
- This protects the RNA at the 5' so ribonucleases can't degrade the RNA


Transcription termination:

- C terminal domain recruits CPSF and CstF
- Move from C terminal domain onto the nascent RNA molecule
- Cleave the RNA 3' downstream and CstF drops off
- Cleaved RNA recruits polyA pol which only adds A's to the 3' end.
- The tail of A is recognised by the polyA binding protein
- It is further stabilised when bound by polyA binding proteins


Where does tail length modification occur?

The cytoplasm!


Rat1 and hXrn2 are RNase proteins. What do they do?:

- The right polyadenylation signal causes de-capping of the end
- RNase degrades it
- Depends on genes
- The Polymerase falls off


Splicing relies on three conserved sites:

- Cuts out introns which can be very complex structures
- 5' end of the intron has a conserved dimer of nucleotides as a splice site
- 3' end has a tri-nucleotide pyrimidine splice site
- Lariat signal in the middle


The lariat site creates a loop:

- 5' end of the intron is covalently joined to the inside of the intron
- The lariat is degraded and the spliced form of the RNA is the produce


Transcript splicing and recognises signals:

- Spliceosomal machinery catalyses intron splicing
- Complex consists of 150 proteins, 5 smaller nuclear RNAs, snRNAs (which provide specificity) associated with proteins to form snRNPs
- snRNPs bind in succesion to pre-mNRA



- Recognise the 5' splice site and branch site
- Bring together the 5' splice site and the branch site
- Catalyse RNA cleavage and joining reactions


Splicing errors result in errors in translation:

- eg) Thalassemia diseases
- Cryptic splice signals can randomly occur and misidentification can result in incorrect protein products
- Proteins can interact with introns and exons to help find and assist in accurate splicing


Other types of splicing..

- The major splice mechanism is U1 recognising 5' splice signal, U4 recognising branch site etc.
- Some introns have an AU instead of a GU so they use a less abundant snurp for the splice signal and the branch site.
- Trans-splicing is another method of splicing!



- Recognition of 5' sequence, recruits U4, 6 and 5
- On a different RNA U2 is bound at the branch site
- Associating results in these two exons from different RNA molecules binding


Why is trans-splicing useful?

- It allows a common region to be attached.
- In C. elegans mRNAs are trans-spliced to attach a 5' leader sequence
- Polycistronic re-mRNAs are cleaves, cis-spliced and trans-spliced
- eg) adding a secreting sequence to allow movement from the cell into the external environment in tropanosomes