A molecular genetic approach will be used to elucidate the mechanisms of action of a complex set of interacting regulators of gene expression. The bacteriophage P22 antirepressor gene, ant, is regulated at the transcriptional level by two repressors, the products of the linked arc and mnt genes, and at a post- transcriptional level by a small antisense RNA, sar, which is complementary to the ant ribosome binding site. Post-transcriptional control of ant by sar RNA will be investigated further to elucidate the mechanism by which the antisense RNA represses synthesis of antirepressor. RNA's produced in vivo will be analyzed to determine whether sar RNA affects ant mRNA stability and how repression depends on the abundance of the two RNA's. The possible involvement of a protein in pairing of RNA's will be investigated. Mutations in the RNA's that affect the pairing reaction and mutations in the bacterial host that affect the efficiency of antisense RNA control will be sought and characterized. Experiments are planned to elucidate the mechanism by which an unusual polar mutation interferes with ant gene expression. This mutation may potentiate premature termination of transcripts or create an unfavorable mRNA secondary structure that interferes with translation of ant or increases the rate of mRNA degradation. Other unusual mutations affecting antirepressor synthesis will be sought. The kinetics of synthesis of Mnt repressor protein and mnt mRNA will be measured with various P22 mutants to corroborate evidence that the ant and mnt promoters compete for polymerase, that Mnt protein stimulates its own synthesis, and that Arc protein represses synthesis of Mnt protein. This analysis will also determine whether the promoter for sar RNA also serves as a promoter for mnt expression during establishment of lysogeny by P22. The effect of temperature on Mnt synthesis will be investigated and a mutation that may effect mnt expression will be analyzed.
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