​Given below is the structure of a gene whose transcription is terminated in a Rho-independent manner. When the terminator is operational, the short transcript of 150 bases is formed. When it is not operational, a longer transcript of 200 bases is produced. A researcher generated mutations in the terminator region and examined the transcript lengths.

The manipulations performed were:

(A) Three nucleotides of the string of 8Ts were replaced by GCC.
(B) The 8T sequence was transferred to the template strand.
(C) The sequences generating the paired stem were altered to disrupt pairing.
(D) The sequences generating the paired stem were altered to disrupt pairing, but compensatory mutations restored pairing.

Which option correctly predicts the transcript sizes in each case?

Correct option is D

Explanation:

Understanding Rho-independent Termination Mechanism:

• Rho-independent termination relies on two key features:
1. GC-rich stem-loop structure (hairpin) in the mRNA.
2. A string of uracils (U) following the hairpin.
• Mechanism:

• The hairpin structure causes RNA polymerase to pause.
• The weak U-A interactions (between the transcript and template DNA) allow RNA to dissociate, ending transcription.
• Disrupting either the hairpin or the U-rich sequence can prevent termination, leading to a longer transcript.

Analysis of Each Mutation:

(A) Three nucleotides of the string of 8Ts were replaced by GCC.

• Effect on termination:
• The string of uracils is crucial for proper termination because U-A bonds are weak.
• Replacing three uracils with G/C bases strengthens base pairing, making termination inefficient.
• Expected transcript size:
• Long transcript (200 bases) because termination is disrupted.

(B) The 8T sequence was transferred to the template strand.

• Effect on termination:
• Normally, Ts in the non-template strand result in U’s in the mRNA transcript.
• If Ts are moved to the template strand, the transcript will have A’s instead of U’s.
• A-rich sequences do not cause termination, as A-U pairs are stronger than U-A pairs.
• Expected transcript size:
• Long transcript (200 bases) because the U-run is missing, preventing termination.

(C) Disrupting the hairpin structure.

• Effect on termination:
• The hairpin structure is necessary to pause RNA polymerase.
• If hairpin formation is disrupted, termination efficiency drops significantly.
• Expected transcript size:
• Long transcript (200 bases) because RNA polymerase fails to pause.

(D) Disrupting the hairpin but restoring it with compensatory mutations.

• Effect on termination:
• Compensatory mutations restore base pairing in the hairpin.
• Since the hairpin functions properly, termination will occur as expected.
• Expected transcript size:
• Short transcript (150 bases) because termination is restored.

Information Booster:

1. Rho-independent termination depends on the formation of a GC-rich hairpin followed by U-rich sequences.
2. U-A interactions are weaker than G-C interactions, making the termination site susceptible to disruption.
3. Mutation in the U-run region can lead to transcription readthrough, increasing transcript length.
4. Disrupting the hairpin structure prevents RNA polymerase pausing, causing failure in termination.
5. Compensatory mutations restoring the hairpin structure can recover proper termination efficiency.
6. Mutations in bacterial terminators can lead to antibiotic resistance by altering gene expression levels.

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