Northern Illinois University, Biological Sciences, Dekalb, IL 60115
pilE gene encodes the main component, PilE polypeptide, of the pilus structure in N. gonorrhoeae. Transcription of pilE is enhanced by integration host factor (IHF), a small DNA binding protein that acts as a transcriptional cofactor (Hill, 1997). When the IHF binding site was deleted the level of pilE mRNA decreased dramatically, while significant amount of small pil-specific RNA was observed. Using RNA secondary algorithms the 5’ pilE untranslated region (5’UTR) was predicted to form three stable loop structures with loop 3 containing the ribosome binding site (rbs). When the predicted loops 1 or 2 were deleted from translational fusions, using pilE rbs and a reporter gene (beta galactosidase or chloramphenicol acetyl transferase (CAT)), a significant decrease in transcript level compared to the intact pilE 5’ UTR was observed using quantitative real-time PCR (qRT PCR) in conjunction with chemical reactions (i.e. beta galactosidase assay and CAT assay). This implies that these secondary structures play a role in maintaining the stability of pilE transcript. Transcriptional fusions with rbs from the CAT gene were then constructed using various pilE promoters in which each of the three loops was deleted. In E. coli, the mRNA was again unstable yet all the transcriptional fusion clones were all chloramphenicol resistant regardless of loop deletions. In contrast, translational fusions in which either loop 1 or loop 2 was deleted yielded very little CAT protein compared to the wild-type, implying that the pilE rbs within loop 3 may be occluded in certain conditions. We then explored the possibility that the putative stem loop structures are involved in an anti-sense model for pilE expression. qRT PCR analysis as well as Northern blot using a pilE anti-sense strand-specific probe indicated the presence of cis-anti-sense RNA. Additionally, an anti-sense promoter element located at the 3’ end of pilE was identified. When this promoter was mutated, transcript level decreased approximately 10-fold. However, in gonococci containing these loop-deleting constructs, mutation of this anti-sense promoter did not affect expression of the pilE gene. This suggests that the cis-anti-sense RNA does not play a regulatory role in pilE gene expression. Investigation into other potential roles of this anti-sense RNA is ongoing.
[Abstract (DOCX)]