Caulobacter crescentus is an ±-proteobacterium that goes through distinct morphological changes during its cell cycle, making it an excellent model for the study of morphology and gene regulation. Under stress, such as cold-shock, C. crescentus adapts its metabolism to maintain its viability, involving changes in gene expression. On these conditions, RNA molecules tend to form stable secondary structures, impairing their translation and degradation. RNA helicases are proteins with the ability to remodel RNA structures and RNA-protein complexes, participating in processes associated with gene expression. The DEAD-box family of RNA helicases is the biggest and the most studied in bacteria. Some members of this family are often found in association with a multienzimatic complex called RNA degradosome and participate in several cellular processes such as RNA turnover, ribosome biogenesis and stress response. Studies have shown the importance of one of these helicases (RhlB) to the cell fitness and adaptation to low temperature. The aim of this project is to define the participation of the DEAD-box RNA helicase RhlB in the processing of cellular RNAs and in the ribosome biogenesis in Caulobacter. To achieve this, the RNA profile in the mutant rhlB strain will be identified under different growth conditions to assess the role of this helicase on global gene expression. A strain with FLAG-tagged RhlB will be constructed to evaluate its interaction with specific RNAs, particularly sRNA and mRNAs, and mapping of the specific interaction sites of this helciase with the degradosome will be determined through site-directed mutagenesis. Assays to assess the association of RhlB to the ribosome, as well as its participation in the rRNA 5S maturation will be performed.
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