STUDY OF THE REGULATION OF EXPRESSION OF ACONITASE AND THE EFFECT OF ITS LIGATION TO THE RNA DEGRADOSOME IN CAULOBACTER CRESCENTUS

• Grant number: 24/09014-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: September 01, 2024
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Marilis Do Valle Marques
• Grantee: João Pedro de Carvalho Pereira
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Aconitase is an enzyme that participates in the Krebs Cycle, catalyzing the reversible conversion of citrate to isocitrate. Furthermore, in many other organisms from diverse groups, this protein, in addition to its enzymatic function, also has a regulatory role by binding to certain messenger RNAs and interfering with their stability and translation. In Caulobacter crescentus, it appears that aconitase also possesses this regulatory function; however, in this organism, it is associated with a protein complex called the RNA degradosome, which in prokaryotes carries out RNA turnover. Previous studies have shown that detaching aconitase from the degradosome results in changes in the cellular amount of aconitase and in the stability of its messenger RNA. This project aims to evaluate the impacts that the degradosome's binding has on the regulatory function of aconitase and to understand its transcriptional and post-transcriptional regulation in Caulobacter crescentus. To characterize the regulation of expression, transcriptional and translational fusions of its regulatory region with the reporter gene lacZ will be constructed in the placZ290 and pJBZ281 vectors, respectively, and Beta-galactosidase assays will be conducted under various conditions. To assess the impacts of its binding to the degradosome, a strain in which the aconitase binding site in the degradosome has been removed (RNED675) will be used. We will utilize a strain in which codons for a FLAG tag have been added to the beginning of the aconitase ORF, to enable the purification of the protein with its binding RNAs and sequencing these by RNA-seq (HITS-CLIP). After identification, we will analyze their stability via qRT-PCR in both the wild-type and RNED675 strains.