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

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage h. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tl mutations map in the G+C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tl is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.