An in vitro translation system capable of incorporating ~8 amino acids/s was programmed to translate a single mRNA that codes for an alanine-rich (~35% alanine with uniform distribution of alanine) protein of 275 amino acids (~30 kDa) including a hexa-histidine tag at the C-terminal end of the protein.
The protein possesses three methionine residues at amino acid positions 1, 135 and 230 and generates polypeptides of ~15 kDa, ~10 kDa and ~5 kDa upon degradation with cyanogen bromide.
The translation reaction was initiated and the ongoing reaction was supplemented with ¹⁴C Ala after 5 min. Soon after addition of ¹⁴C Ala, aliquots were drawn at 2, 20, and 200 s, and reactions in the aliquots were instantaneously stopped. The translated proteins were purified on Ni-NTA columns, processed for degradation by CNBr, resolved on SDS-PAGE, and visualized by nonquantitative autoradiography.

Which of the following autoradiograms represents the expected pattern of the bands?

Correct option is C

Explanation-

Given:
A protein coding mRNA of 275 amino acids (~30 kDa) with 3 methionines at - Position 1, Position 135 and Position 230
This protein is alanine-rich (~35%) and tagged with a 6xHis tag at the C-terminal for purification. Translation was initiated in vitro and supplemented with radioactive ¹⁴C-Ala at 5 minutes.
Aliquots were taken at 2s, 20s, 200s, translation stopped, protein purified, and digested with CNBr (cleaves at methionine). Fragments generated by CNBr are ~15 kDa, ~10 kDa, and ~5 kDa.

CNBr (Cyanogen Bromide) cleaves C-terminal to methionine residues.
So a protein with Met at 1, 135, 230 will be cut into:
                                                   Residues 1–134 (~15 kDa)
                                                               135–229 (~10 kDa)
                                                               230–275 (~5 kDa)

Pulse-labeling with ¹⁴C-Ala after 5 min:
                 Labels only newly synthesized proteins.
                 The earlier the timepoint, the less complete translation.
SDS-PAGE shows only ¹⁴C-labeled fragments.
​
By Analyze the Timepoints:
At 2 seconds: Only short peptides are made → the ribosome likely hasn’t gone far beyond the first ~40–50 amino acids.
          After CNBr cleavage, only the first fragment (15 kDa) may show partial labeling, or no fragment fully labeled.
At 20 seconds: Enough time for elongation up to second or third methionine. 
          Expect partial translation of longer fragments.
          Possibly labeling of 10 kDa and 5 kDa regions.
At 200 seconds: Full-length protein synthesized.
          All three CNBr fragments can appear strongly labeled.

Expected Autoradiogram Pattern:
2 sec → Weak labeling, possibly 5 kDa only or none strongly visible.
20 sec → Moderate labeling of 5 kDa, and maybe 10 kDa bands.
200 sec → Strong labeling of all 3 bands (15, 10, 5 kDa).

Option C  is the correct answer because it accurately reflects the time-dependent synthesis and labeling of the CNBr-cleaved protein fragments, as visualized via autoradiography.
2 sec: Faint or single lower band (incomplete protein)
20 sec: Multiple bands (partial labeling of intermediate-length proteins)
200 sec: All bands clearly visible (fully translated and labeled proteins)