Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA...

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• The tI terminator sequence in bacteriophage lambda is located downstream of the int gene.
• tI is known to function as a transcription terminator and may also stabilize the int mRNA.
• The precise roles of tI in transcription termination and RNA stability require further investigation.

Purpose of the Study:

• To investigate the role of the tI sequence in transcription termination.
• To determine the function of tI in maintaining mRNA stability.
• To characterize the effects of tI mutations on these processes.

Main Methods:

• Isolation and characterization of three point mutations (tI1, tI2, tI3) in the tI terminator.
• In vivo and in vitro assays to measure transcriptional termination efficiency (e.g., galactokinase activity, E. coli RNA polymerase assays).
• Analysis of transcript stability in wild-type and polynucleotide phosphorylase-deficient host strains.

Main Results:

• All tI mutations mapped to the G+C-rich dyad symmetry region.
• Mutations reduced in vivo termination from 99% to 81-93% and in vitro termination from 80% to 8-12%.
• tI mutations caused upstream transcript instability in vivo, which was rescued in a polynucleotide phosphorylase-deficient host.

Conclusions:

• The intact hairpin structure of the tI terminator is essential for efficient transcription termination.
• The tI sequence plays a critical role in maintaining mRNA stability.
• tI functions by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.