1887

Abstract

Regulation of the gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. analysis predicts the formation of three putative stable stem–loop structures with favourable free energies within the 5′ untranslated region of the message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem–loop mutations diminishing loop stability. Utilizing a series of translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the transcript from degradation. Putative loop 3 contains the ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the promoter that is predicted to hybridize across the 5′ untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants with the antisense RNA gene facilitates translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating expression levels.

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2016-11-23
2019-12-14
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