Lecture 14

Question 1

Core RNAP complex and GTFs:

Answer 1

• Recognises upstream control sequences (UCS)

- Upstream binding factor (UBF) recognises UCS

Question 2

Ribosomal RNA genes:

Answer 2

• 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)

Question 3

RNAPIII:

Answer 3

• 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

Question 4

RNAPII

Answer 4

• 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

Question 5

The primary transcript is processed in the nucleus:

Answer 5

• Capping
• Splicing
• Polyadenylation and termination

Question 6

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

Answer 6

• 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.

Question 7

Capping process:

Answer 7

• 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

Question 8

Transcription termination:

Answer 8

• 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

Question 9

Where does tail length modification occur?

Answer 9

The cytoplasm!

Question 10

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

Answer 10

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

Question 11

Splicing relies on three conserved sites:

Answer 11

• 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

Question 12

The lariat site creates a loop:

Answer 12

• 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

Question 13

Transcript splicing and recognises signals:

Answer 13

• 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

Question 14

Snurps:

Answer 14

• Recognise the 5’ splice site and branch site
• Bring together the 5’ splice site and the branch site
• Catalyse RNA cleavage and joining reactions

Question 15

Splicing errors result in errors in translation:

Answer 15

• 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

Question 16

Other types of splicing..

Answer 16

• 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!

Question 17

Trans-splicing:

Answer 17

• 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

Question 18

Why is trans-splicing useful?

Answer 18

• 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