3'-end formation at the phage lambda tR1 rho-dependent transcription termination site

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• The rho-dependent transcription terminator tR1 in bacteriophage lambda regulates RNA synthesis downstream of the major rightward promoter (PR).
• Discrepancies exist between in vitro and in vivo observed RNA termination sites for lambda PR transcription.

Purpose of the Study:

• To reconcile the differences in RNA termination sites observed in vitro versus in vivo for bacteriophage lambda PR transcription.
• To analyze and compare lambda PR transcription products across purified in vitro systems, S30 cell extracts, and in vivo conditions.

Main Methods:

• Comparative analysis of RNA products from purified in vitro transcription, translationally active S30 cell extracts, and in vivo samples.
• RNA characterization using S1 nuclease mapping to identify 5' and 3' termini.
• Incubation of in vitro transcripts in S30 cell extracts to observe degradation and processing.

Main Results:

• In vitro transcription yielded heterodisperse 3' termini, while in vivo and S30 extracts showed more defined termination/processing sites at +263 and +311 nucleotides.
• S30 cell extracts produced transcripts terminating at +263, +308, and +318, with +263 and +308 identified as potential RNA processing sites.
• In vitro transcripts incubated in S30 extracts were degraded to products of 263 and 308-311 nucleotides.

Conclusions:

• Cellular components, particularly in S30 extracts, play a crucial role in determining specific RNA termini and processing, explaining the in vitro-in vivo discrepancy.
• RNA processing at +263 and +308/311 significantly influences the observed termination patterns in cellular environments.
• The tR1 terminator's activity is modulated by cellular factors, leading to distinct transcript profiles in vitro versus in vivo.