Question

11. What are the functions of the 5’ cap? How is it added on the RNA?

12.Explain how a poly-A tail is produced; (CPSF and CstF, Poly APolymerase, Poly A Binding Protein (PABP)).

13.What are the two hypotheses for eukaryotic transcription termination?

14.How does splicing of pre-mRNA occur?

15.Compare and contrast splicing of group I introns, group II introns and spliceosome-mediated splicing of mRNA.

Answer 1

11. Functions of 5' Cap

a. Protection of mRNA from degradation

b. transport of the mRNA from nucleus to cytoplasm

c. binding of ribosome with mRNA

Euaryotic mRNA has a peculiar enzyatically appended cap structure consisting of 7-methylguanosine residue joined via 5'-5' triphosphate bridge. During Transcription, 7-mG is added to 5' end of nascent mRNA. The initial steps in RNA capping are catalyzed by a dimeric capping enzyme which associates with the phosphorylated carboxy terminal tail domain of RNA pol II. One subunit of the capping enzyme removes gamma phosphate from 5' end of the nasecnt RNA emerging from the surface of an RNA pol II. The other subunut transfers the GMP moiety from the GTP to the 5'dipohosphate of the nascent transcript, creating the guanosine 5'-5' triphosphate structure. In the final steps, separate enzymes transfer methyl groups form S-adenosylmethionine to N⁷ position of the guanine at the 5' end of the nascent RNA. If mRNA has methyl group on N7 position then it is called cap0. this is the first methylation step and occurs in all eukaryotes.

12. A single processing complex undertakes both the cutting and polyadenylation. The signal is needed for both cleavage and polyadenylation. In mammals,polyadenylation is directed by a signal sequence in the mRNA almost invariably 5'-AAUAAA-3'. The sequence is located between 10 to 30 nucleotides upstream of the polyadenylation site, which is often immediately after the dinucleotide 5'-CA-3'. 10-20 nucleotides downstream of the polyadenylation site is a GU-rich region. Both the poly(A) signal sequence and teh GU rich region are binding sites for multi subunit protein complexes which are respectively the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF).. Generation of the proper 3' terminal structure requires an endonuclease (consisting of the components CFI, cleavage factors I and CFII, cleavage factors II) to cleave the RNA. Cleavage is followed by polyadenylation in a tightly coupled manner. The poly (A) polymerase catalyzes the polyadenylation reaction in template independent manner. Its substrate is ATP. The polyadenylation reaction passes through two stages. First, a short oligo sequence is added to 3' end. In the second phase the oligo (A) tail is extended to full 200 residue length. The reaction requires another stimulatory factor that recognizes the oligo (A) tail. This additional factor included Poly-adenylate binding portein (PABP) which helps the polymerase to add the adenosines possible also influence the length of the poly A tail.

13. For Pol I genes, transcription is stopped using a terminator factor similar to a rho-dependent mechanism. For transcription termination pol III genes, it ends with a termination sequence that includes a polyuracel stretch, by a mechanism resembling rho-independent prokaryotic termination. Termination of Pol II is functionally coupled with RNA processing in which the 3' end undergoes modification

14. There are two types of pre mRNA introns-GU-AG and AU-AC where the former is more common. In GU-AG inton the first two nucleotides of the intron sequence are 5'GU-3' and the last two 5'-AG-3' (3' splice site or acceptor site). A branch point adenosine is also present and is usually located 20-50 bases from 3' splice site. A polypyrimidine tract is also present near the 3' end. Splicing includes two transestrification reactions. The first cleaves the 5' splice site. The hydroxyl on 2'C of the adenosine on the branch point attacks the phosphodiester bond of 5'G, leaving a free 3'hydroxyl group. This leads to the formation of a new 5'-2' phosphodiester bond linking the first nucleotide of the intron with internal adenosine (This forms a lariat structure). The 3' hydroxyl group now created where the phosphodiester bond was broken, attacks the phosphodiester bond at the 3' splice site in the second transestrification reaction. After this, the intron leaves and the exons are joined together.