Post Transcription

Question 1

mRNA structure in eukaryotes

Answer 1

• Eukaryote mRNA contains a 7-methyl guanine cap on the 5 end and polyadenylation tail at the 3 end

Question 2

mRNA structure in prokes

Answer 2

• The 5 and 3 ends of bacterial mRNA are unmodified
• mRNA polycistronic
  
  • ​encode for multiple proteins on the same mRNA

Question 3

what does RNA pol II carry?

Answer 3

• Carries a set of pre-mRNA processing proteins on its tail
• Shortly following the initiation of transcription these proteins are transferred to the nascent RNA
• This transfer of RNA processing proteins typically occurs after ~25 nucleotides are produced

Question 4

describe the capping process of mRNA (4 enzymes)

Answer 4

1. Phosphatase: removes 1 phosphate from the 5 end of the RNA
2. Guanylyl transferase: adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’)
3. Guanine-7-methyl transferase: adds a methyl group to the 7 position of the terminal guanine
4. 2’O-methyl transferase: adds a methyl group to the 2’-O position to the next to last base on the 5’end

Question 5

what are the 3 functions of 5’ mRNA capping

Answer 5

1. Regulates export of mRNA out of the nucleus
   
   1. mRNA is exported in complexes that contain a Cap Binding Complex (CBC) at the 5’ end and RNA binding proteins along the rest of sequence
2. Required for the efficient translation of the mRNA into protein
   
   1. The CBC is replaced by translation factors which facilitates binding of mRNA to the ribosome
3. Prevents 5’ degradation
   
   1. The CBC and elF block the access of de-capping enzymes to the cap. Increase half life of mRNA

Question 6

name the enzymes invovled in the 3’ mRNA polyadenylation in Euks

Answer 6

1. Cleavage and polyadenylation specificity factor (CPSF) binds to the polyadenylation signal
2. Cleavage stimulating factor F (CstF) binds the GU-rich element beyond the cleavage site
3. Cleavage factors bind to the CA sequence at the cleavage site

Question 7

describe the polyadenylation sequence

Answer 7

• Poly-A-polymerase (PAP) adds 200 A nucleotides to the 3’ end produced by the cleavage
• Poly-A Binding Protieins (PABP) binds to the poly-A tail and assist in directing translation by the ribosome
• The cleaved fragment of the RNA is degraded in the nucleus

Question 8

name the 4 functions of 3’ mRNA polyadenylation

Answer 8

1. Increases the half life of the mRNA by protecting it from enzymatic degradation in the cytoplasm
   
   1. PolyA-binding protein (PABP) binds to tail protecting mRNAs from ribonuclease attack
2. PABP interacts with e-IF 4G and this interaction leads to stimulation of translation of euk mRNAs
3. Aids in transcription termination
4. Aids in export of the mRNA from the nucleus

Question 9

describe the structure of introns

Answer 9

1. Splice donor site (at the beginning): GU
2. Conserved Branch Point Sequence (in the middle): A
3. Splice acceptor site (at end): AG

Question 10

steps of intron removal by spliceosome

Answer 10

1. U1 snRNP binds to the 5’ splice junctions and U2 binds to the branch point
2. Interactions with U4/U6 snRNP and U5 snRNP binds U1 and U2 together to form a loop
3. U4 dissociates forming the active spliceosome
4. The spliceosome complex cleave the intron from exon 1 at the 5’ splice junction. The free end binds to the A in the branch point sequence (forms lariat)
5. The intron is excised by cleavage at the 3’ splice junction and exons 1 and 2 are ligated. snRNPs released.

Question 11

what clinical relevance does problems with spliceosome have?

Answer 11

• Systemic Lupus Erythematosis
  
  • Autoimmune disease with multiple symptoms such as fatigue, arthritis, fever, skin rashes and kidney problems
• Individuals with this condition possess antibodies that react with the U1 RNA component of the spliceosome and various other nuclear proteins such as histone and topoisomerases

Question 12

how is alternative RNA splicing controlled?

Answer 12

• Alternative splicing can occur due to intron sequence ambiguity
  
  • Due to “weak” splice site sequences and therefore splicing choice occurs by chance
• Directed by negative and positive control via proteins that bind to splice sequences and either repress of activate splicing at that site.

Question 13

mutations that destroy or generate new splice sites

Answer 13

• B-thalassemia
  
  • Individuals with B-T have mutations in the B-globin gene that generate additional splice site within the mRNA
  • As a result, frame shifts or premature stop codons are introduced into the mature mRNA
    
    • mRNA usually longer than normal
  • Results in production of an abnormal B-globin protein in hemoglobin
    
    • can cause severe anemia

Question 14

RNA editing example (liver vs. intestine)

Answer 14

1. In mammals, the apo-B gene encodes 2 alternative forms of the apolipoprotein B
   
   1. In the intestine, the apo-B mRNA is edited so that a premature stop codon is produced (CAA –> UAA) leading to the synthesis of the shorter Apo-protein
   2. The editing is accomplished by a cytidine deaminase enzyme

Question 15

RNA editing example (glutamate receptor)

Answer 15

• Editing of the glutamate receptor involves a single base change from A to I (inosine)
  
  • This editing results in a codon change within the mRNA from glutamine to arginine
• The editing affects an amino acid located in the inner wall of the ion channel and alters Ca2+ permeability. This editing is a requirement for proper brain development.
  
  • Accomplished by the enzyme adenosine deaminase

Question 16

RNA editing in parasites?

Answer 16

• Trypanosomes (Chagas Disease) and Leishmania (leishmaniasis) editing is more extensive and involves complementary base pairing by a “guide RNA”.
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