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| Author(s): |
Rodolfo Alvarenga Ribeiro
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2018-12-05 |
| Examining board members: |
Marilis do Valle Marques;
Tie Koide;
Julio Cezar Franco de Oliveira;
Robson Francisco de Souza
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| Advisor: | Marilis do Valle Marques |
| Abstract | |
DEAD-box RNA helicases (DEAD-box RNA helicases) are enzymes involved in several important biological processes, such as the degradation and stabilization of nucleic acids, assembly of riboprotein complexes, and biogenesis of the ribosome. Caulobacter crescentus is an oligotrophic bacterium belonging to the α-proteobacteria group, which has three genes encoding RNA helicases of the DEAD-box family. Previous results suggest that one of these enzymes, DbpA, encoded by the CC1478 gene, is a functional helicase RNA, but has a poorly conserved carboxy-terminal end, with possible ribosome anchoring function. This project aims to characterize the regulation of DbpA, defining its regulation, to evaluate the phylogenetic information to determine its evolutionary origin, and to determine the importance of this C-terminal domain for the function of the enzyme. Protein expression in response to stress, and its interaction with protein complexes, such as the ribosome, were also evaluated. In order to characterize the function and regulation of the CC1478 gene, a strain was obtained where the protein contained an amino-terminal tag. Expression at the transcriptional level was measured by qRT-PCR and at the translational level by Western blotting, and little difference was observed for protein levels in the various stresses. Results obtained with the truncated constructs of the C-terminus indicate that the protein lacking the C-terminal extension loses part of its function compared to the wild type, and that the DbpA domain is important for its function. Analysis of the interaction with the ribosomal subunits showed that the protein was present in the ribosome subunits as well as in complete ribosomes fractions. Phylogenetically the C-terminal tail is a hyper-variable region, rich in charged amino acids, but not always in the same amount. In the evolutionary aspect this family of proteins is rich in paralogues, and many are still complementary in their function. The RRM (RNA-recognitiion-motif) clan, in which DbpA is included, has an origin as basal as LUCA, suggesting that these proteins have always been acting in the assembly in the ribosome, and in redundancy, providing versatility in face of the environmental stresses. (AU) | |
| FAPESP's process: | 16/06378-3 - Characterization of the DEAD-box RNA helicase CC1478 in Caulobacter crescentus and its regulation |
| Grantee: | Rodolfo Alvarenga Ribeiro |
| Support Opportunities: | Scholarships in Brazil - Master |