title: Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.
abstract: The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.
authors.affiliation: Research Service, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA.
authors.forename: James
authors.initials: J
authors.lastname: Matsunaga
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Schlax
authors.forename: David A
authors.initials: DA
authors.lastname: Haake
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics / Q000528:radiation effects*; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D019677:Artificial Gene Fusion; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000528:radiation effects; D015964:Gene Expression Regulation, Bacterial / Q000528:radiation effects*; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D007921:Leptospira interrogans / Q000235:genetics / Q000528:radiation effects*; D009690:Nucleic Acid Conformation; D013696:Temperature; D015533:Transcriptional Activation / Q000528:radiation effects*
chemical_list: D020121:5' Untranslated Regions; D000942:Antigens, Bacterial; C487005:LigA-m protein, Leptospira interrogans; C487006:LigB-m protein, Leptospira interrogans