please answer all the questions • Post-transcriptional Processing of Eukaryotic
ID: 196635 • Letter: P
Question
please answer all the questions
• Post-transcriptional Processing of Eukaryotic mRNA o List the four enzymes that comprise the Cap Synthesizing Complex.
o What is the function of each enzyme in the attachment of the 7-methylguanosine cap. RNA triphosphatase Guanylyltransferase (capping enzyme) Guanine-7-methyl-transferase.
o Where does the Cap Synthesizing Complex bind? When does capping of mRNA take place? What is the function of cap binding complex (CBC)?
o How is mRNA processed by 2’-O-methyltransferase? Why might this prove to be beneficial?
o What is the function of the following proteins in the process of adding a polyA tail to the 3’- end of mRNA and where do they bind? CPSF PAP PABP
Explanation / Answer
There are three enzymes that comprise the Cap Synthesizing Complex, the RNA triphosphatase, guanylyltransferase and methyltransferase. Function of these enzymes:-
RNA triphosphatase removes the terminal phosphate group from the 5' end.
mRNA guanylyltransferase adds a GTP molecule to the 5' bisphosphate losing a pyrophosphate from the GTP substrate in the process.
The enzyme (guanine-N7-)-methyltransferase transfers a methyl group from S-adenosyl methionine to the guanine ring.
The cap synthesizing complex binds to the RNA polymerase II initially before transcription starts. As soon as the 5' end of the new mRNA emerges out from the RNA polymerase the capping complex carries on with the capping process. The CBCs help in the export of the mRNA and protect it from decapping. They also serve as a marker for the pioneer round of translation when the message is examined by nonsense mediated decay.
mRNA Cap 2´-O-Methyltransferase adds a methyl group at the 2´-O position of the first nucleotide adjacent to the cap structure at the 5´ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (cap-0) resulting in a cap-1 structure. The cap-1 structure has been reported to enhance mRNA translation efficiency and hence may help improve expression in mRNA transfection and microinjection experiments.
CPSF or Cleavage and polyadenylation specificity factor is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (pre-mRNA) molecule in the process of gene transcription. It is the first protein to bind to the signaling region near the cleavage site of the pre-mRNA. The upstream signaling region has the canonical nucleotide sequence AAUAAA, which is highly conserved across the vast majority of pre-mRNAs. A second downstream signaling region, located on the portion of the pre-mRNA that is cleaved before polyadenylation, consists of a GU-rich region required for efficient processing.
PAP or Poly(A) Polymerase(polynucleotide adenylytransferase) is the enzyme responsible for adding a polyadenine tail to the 3' end of a nascent pre-mRNA transcript. Its substrates are ATP and RNA.
Poly(A)-binding protein (PABP) is a RNA-binding protein which binds to the poly(A) tail of mRNA. The binding protein is also involved in mRNA precursors by helping polyadenylate polymerase add the poly(A) nucleotide tail to the pre-mRNA before translation. PABP binds to the A-rich UTR region at the 3' end of mRNA.
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