8. An eukaryotic cell undergoing mRNA synthesis and processing was incubated with³²P labelled ATP, with the label at the β-position.
Where do you think the radioactive isotope will appear in the mature mRNA?
(1) ³²P will not appear in the mature mRNA under any circumstances because β and γ phosphates are released during transcription.
(2) Phosphate groups of the phosho-diester backbone of the mRNA will be uniformly labelled as only γ phosphates are released during transcription.
(3) ³²P will appear at the 5′ end of the mRNA if only it has “A” as the first nucleotide.
(4) No ³²p will appear in the mature mRNA because the 5′-terminal phosphate of an “A” residue will be further removed during the capping process.

Where Does β-32P-Labeled ATP Appear in Mature Eukaryotic mRNA?

Correct Answer:

(3) 32P will appear at the 5′ end of the mRNA if only it has “A” as the first nucleotide.

Detailed Explanation:

• During transcription, RNA polymerase incorporates nucleoside triphosphates (NTPs) into the growing RNA chain by linking the α-phosphate of the incoming NTP to the 3′ hydroxyl group of the preceding nucleotide, releasing the β and γ phosphates as pyrophosphate.

• When ATP labeled at the β-phosphate is used, the β-phosphate is normally released during polymerization, so the radioactive label does not become part of the RNA backbone.

• However, the 5′ end of the nascent RNA retains the original triphosphate group of the first nucleotide, which includes the β-phosphate. If the first nucleotide incorporated is adenosine (A), the β-32P label on ATP will remain at the 5′ end of the mature mRNA.

• This is because the 5′ end of the RNA is initially a triphosphate, and the β-phosphate from the initiating ATP remains attached until the capping process modifies the 5′ end.

• If the first nucleotide is not “A,” the β-32P label from ATP will not be incorporated at the 5′ end.

Why Other Options Are Incorrect:

1. 32P will not appear in mature mRNA under any circumstances because β and γ phosphates are released during transcription.
   Incorrect. The β-phosphate of the first nucleotide remains at the 5′ end if that nucleotide is ATP.

2. Phosphate groups of the phosphodiester backbone will be uniformly labeled as only γ phosphates are released.
   Incorrect. The α-phosphate of each NTP is incorporated into the backbone; β and γ phosphates are released, so β-labeled ATP does not label the backbone uniformly.

3. No 32P will appear because the 5′-terminal phosphate of an “A” residue is removed during capping.
   Incorrect. The capping process modifies the 5′ end but does not remove the β-phosphate; instead, it forms a unique 5′–5′ triphosphate linkage preserving the β-phosphate.

Summary Table

Conclusion

When ATP labeled at the β-phosphate is used, the radioactive label will appear only at the 5′ end of the mature mRNA if the first nucleotide incorporated is adenosine (A). This is because the β-phosphate of the initiating ATP remains attached at the 5′ end during transcription and capping.