Transcripts that increase the processivity and elongation rate of RNA polymerase

Area of Science:

• Molecular Biology
• Virology
• Gene Regulation

Background:

• Lambdoid phage HK022 utilizes cis-acting antitermination sites (put sites) to overcome transcription termination signals.
• Antitermination is crucial for phage gene expression but often involves complex protein machinery.
• The precise mechanism of HK022 antitermination and its reliance on RNA structure were not fully elucidated.

Purpose of the Study:

• To investigate the mechanism of antitermination mediated by the put sites of phage HK022.
• To determine the role of RNA conformation and cellular proteins in this process.
• To characterize the effect of put sites on RNA polymerase elongation and pausing.

Main Methods:

• Analysis of put mutations affecting predicted RNA stem formation and their impact on antitermination.
• In vitro transcription assays using purified RNA polymerase, template DNA, and substrates.
• Site-directed mutagenesis of the RNA polymerase beta' subunit to assess its role in antitermination.

Main Results:

• Antitermination activity strictly depends on the formation of specific RNA stem structures within the put transcripts.
• Put-dependent readthrough of terminators and increased elongation rates were observed in a purified system, indicating no requirement for additional proteins.
• A mutation in the beta' subunit's zinc-finger region abolished the put-mediated effects on transcription.

Conclusions:

• HK022 antitermination is a simple, RNA-based mechanism that does not require accessory proteins.
• The put transcript's conformation directly modulates RNA polymerase elongation properties, including readthrough and suppression of pausing.
• This RNA-centric mechanism offers a distinct alternative to other known antitermination pathways.