Sequence-specific Rho-RNA interactions in transcription termination

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• The bacteriophage lambda tR1 terminator is crucial for regulating gene expression.
• Termination factor Rho recognizes specific RNA sequences within the nascent cro transcript.
• Understanding ribonucleotide-protein interactions is key to elucidating termination mechanisms.

Purpose of the Study:

• To identify specific ribonucleotide-protein interactions essential for efficient Rho-dependent termination at the tR1 terminator.
• To pinpoint critical nucleotide residues within the tR1 terminator's target region.

Main Methods:

• Construction of a reporter gene plasmid library with single-nucleotide substitutions in the tR1 terminator region.
• Screening of over 16,000 bacterial transformants to identify terminator mutants.
• In vitro transcription assays using purified RNA polymerase and cro gene templates to assess Rho-dependent termination.

Main Results:

• 110 terminator mutants were identified, with most containing multiple substitutions.
• 11 specific single-nucleotide substitutions at eight positions significantly reduced tR1 termination efficiency.
• Approximately half of these critical substitutions also impaired Rho-dependent termination in vitro, highlighting specific residue importance.

Conclusions:

• Specific ribonucleotides within the tR1 transcript are critical for highly efficient Rho-dependent termination.
• While Rho can recognize varied sequences, specific nucleotide interactions enhance termination efficiency.
• Some termination defects may involve interactions with additional cellular factors beyond Rho.