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Restoration of polarity by N-deficiency in lambda phage containing a translocated trp operon segment
Abstract:
When translation of trp mRNA is terminated by a nonsense codon or by antibiotics like chloramphenicol, the amount of the mRNA distal to the blocked ribosomes is found at much lower levels ("polarity"). Polarity is alleviated when the trp mRNA is formed as part of a long transcript from the phage lambda promoter PL (Segawa and Imamoto, 1974; Franklin, 1974); but the relief of polarity is itself largely dependent on the lambda protein N. In a phage that joins the trp operon segment (trpD, C, B A) to a point distal to the N gene, lacking the tL site, synthesis of trp mRNA starting at the PL promoter continues even when translation is generally inhibited by chloramphenicol, but in the absence of functional N gene product synthesis of the mRNA can be blocked by the antibiotic. Unexpectedly, in the absence of N function, even when translation is occurring, weak termination of transcription occurs at some sites in the translocated trp operon.
Insights
Bacterial transcription-translation coupling, known as polarity, is reduced by phage lambda protein N. Even without translation, N-deficient systems show transcription termination in the trp operon.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- Polarity describes reduced mRNA levels distal to translation blocks.
- This phenomenon is often observed with nonsense codons or antibiotics like chloramphenicol.
- Polarity can be alleviated by forming trp mRNA from a longer transcript initiated at the phage lambda promoter PL.
Purpose of the Study:
- To investigate the role of phage lambda protein N in alleviating polarity.
- To understand the mechanisms regulating trp mRNA synthesis and stability.
- To examine transcription termination in the absence of functional N protein.
Main Methods:
- Utilizing phage lambda promoter PL to initiate transcription of the trp operon.
- Employing chloramphenicol to inhibit translation and assess mRNA levels.
- Analyzing trp mRNA synthesis and termination in the presence and absence of functional N gene product.
Main Results:
- Phage lambda protein N is crucial for relieving polarity in trp mRNA.
- In the absence of N function, trp mRNA synthesis can be blocked by chloramphenicol.
- Unexpectedly, weak transcription termination occurs within the translocated trp operon even during active translation when N is absent.
Conclusions:
- Phage lambda protein N plays a significant role in maintaining trp mRNA integrity and preventing premature transcription termination.
- The absence of N protein leads to novel transcription termination events in the trp operon, independent of translation status.
- These findings shed light on the complex regulation of gene expression in prokaryotes, particularly concerning transcription-translation coupling and phage-host interactions.