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Master's Dissertation
DOI
10.11606/D.42.2017.tde-17052017-143528
Document
Author
Full name
Alexandre Magno Vicente
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Marques, Marilis do Valle (President)
Martinez, Cristina Elisa Alvarez
Oliveira, Carla Columbano de
Title in Portuguese
Estudo da RNA helicase DEAD-box codificada pelo gene CC0835 em Caulobacter crescentus.
Keywords in Portuguese
Caulobacter crescentus
Biologia molecular
Microbiologia
Regulação gênica
Resposta bacteriana a estresses
RNA helicases da família DEAD-box
Abstract in Portuguese
Com a construção da linhagem RlhE fusionada a um epitopo, fomos capazes de investigar o acúmulo da proteína após choque frio, em fase estacionária, durante o ciclo celular de Caulobacter crescentus, sob diferentes condições de estresses e, finalmente, após a adição de cloranfenicol, para o estudo a estabilidade protéica. Foi observado um aumento no acúmulo da proteína após choque frio. Além disso, quando em diferentes condições de estresses, RlhE obteve uma leve indução na presença de NaCl e Sacarose; mas permanaceu constante em fase estacionária e durante o ciclo celular. Finalmente, vimos que a estabilidade de RlhE varia de acordo com a temperatura, tendo um aumento da estabilidade a 10°C. As análises de expressão do gene rhlE foram realizadas por ensaios de atividade de betagalactosidase. Demonstramos que a presença da região 5UTR é importante para a indução, e que rlhE possui, pelo menos em parte, uma regulação pós-transcricional. Uma análise transcriptômica global da linhagem selvagem e mutante para rhlE, após o choque frio, foi realizada por RNAseq, o qual nos auxiliou na identificação de genes envolvidos em diversos processos biológicos. Finalmente, a co-imunoprecipitação e identificação dos RNAs por sequenciamento em larga escala revelou que RlhE interage com 51 mRNAs.
Title in English
Study of the DEAD-box RNA helicase encoded by the gene CC0835 in Caulobacter crescentus.
Keywords in English
Caulobacter crescentus
Bacterial stress response
DEAD-box RNA helicases
Gene regulation
Microbiology
Molecular biology
Abstract in English
Here, we constructed a strain in which the RhIE protein was fusioned to an epitope that allowed the investigation of the protein profile after cold-shock, at stationary phase, during cell cycle of Caulobacter crescentus, under different environmental stresses, and, finally, after chloramphenicol addition to study protein stability. The results showed an increase in protein concentration after cold shock stress. When exposed to different stresses RhlE was slightly induced in the presence of NaCl and Sucrose; but its abundance remained constant at stationary phase and during C. crescentus cell cycle. Lastly, the protein stability varies depending on temperature - increasing at low temperature. Gene expression analysis was performed using beta-galactosidase assays. We showed that the presence of the 5UTR is important for the induction of rhlE, and that RhlE is posttranscriptionally regulated. A global transcriptome analysis was performed using RNAseq after cold shock stress of the wild type strain and null mutant for rhlE, and several genes involved in a wide range of biological process were identified. Finally, High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation revealed interactions of RhlE with 51 mRNAs.
 
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Release Date
2019-05-17
Publishing Date
2017-05-17
 
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