UBC Theses and Dissertations
Cloning and characterization of the genes for polynucleotide phosphorylase and ribosomal protein S15 in Escherichia coli Evans, Sylvia
In order to understand the regulation of polynucleotide phosphorylase (PNPase) in Escherichia coli and elucidate its physiological role, the gene for PNPase (pnp) was cloned and characterized. The orientation of the PNPase gene was determined to be anticlockwise on the E_. coli genetic map by means of mapping Tn5 insertions which resulted in truncated peptides. The PNPase clone also produced a < 12,000 MW protein, now known to be ribosomal protein S15 encoded by rpsO. The DNA sequence covering the major 51 transcription termini and control regions for both genes, the entire coding sequence of S15, and possibly the initial coding sequence of PNPase has been obtained. The DNA sequence of S15 agrees with the published protein sequence (Morinaga et al., 1976) with the exception of an additional histidine codon corresponding to amino acid 45. Transcriptional and translational studies of these genes have been carried out by means of β-galactosidase assays and SI mapping of rpsO-lacZ and pnp-lacZ fusion plasmids. The pnp-lacZ fusions have a high level of β-galactosidase activity. Lack of significant β-galactosidase activity in an rpsO-lacZ fusion suggests that S15, like other core ribosomal proteins, may repress its own translation. This is supported by SI mapping results which indicate that the rpsO promoter is active in the rpsO-lacZ fusion. Sequences of the rpsO DNA show some homology to the region of 16S rRNA which binds S15. Mapping results with nuclease SI indicate that the major transcription initiation site for S15 is approximately 100 bp upstream of its AUG start, while that of pnp is approximately 140 bp upstream of its putative AUG start. The major termination event of rpsO transcription occurs approximately 40 bp after the UAA stop codon for S15. A secondary termination event occurs 93 bp distal to this. Evidence from deletion plasmids (Portier, 1982) and Tn5 insertions which result in the disappearance of PNPase protein suggest that the control regions characterized are indeed those for pnp. However, the possibility that the protein fusion thought to be pnp-lacZ is actually the fusion of a small peptide, immediately preceding pnp, to lacZ cannot be excluded.
Item Citations and Data