RNA polymerase is the central enzyme in all gene expression. The rpoCY75N mutation in the zinc-binding domain of the β’ subunit of E. coli RNA polymerase blocks a unique RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022 and its relatives. Here, we describe the characterization of mutant phage, orc0368, which overcomes the rpoCY75N mutation. The orc0368 genome varies from the wild type phage genome by 4 single base pair mutations. Three of these mutations were not characterized because they occur in intergenic regions but the fourth was chosen for study because of its location between a series of transcription terminators and the phage late genes. This mutation was predicted to create a promoter that could potentially drive the expression of gene Q, the late gene regulator. This prediction was confirmed by cloning the respective regions from the wild type and orc0368 phages into a promoter probe vector. Assays of reporter gene activity showed that the sequence originating from orc0368 had significant promoter activity when compared to the wild type sequence. We suggest that the newly created promoter facilitates the expression of phage genes essential for growth on the rpoCY75N strain. However, the small plaque phenotype of orc0368 observed on the mutant host suggests that suppression of the host rpoCY75N mutation is incomplete.
Advisor(s) or Committee Chair
Dr. Rodney King, Dr. Claire Rinehart, Dr. Christopher Keller
Biotechnology | Cell Biology | Genetics | Virology
Ronkainen, Millicent, "Restoration of Phage Growth on a Non-Permissive Host by Bypassing Transcription Termination Signals" (2018). Honors College Capstone Experience/Thesis Projects. Paper 732.