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The contribution of the Hfq protein to interactions of bacterial noncoding RNAs with the mRNA coding sequence

Zuzanna Wróblewska

Abstract

In the presented PhD thesis the contribution of Hfq protein to the annealing of small noncoding RNAs (sRNAs) to the complementary sequences in a target mRNA was analyzed. So far, the detailed molecular mechanism used by Hfq has been described only for its role in the positive regulation of E. coli rpoS mRNA by DsrA sRNA, which recognizes a sequence located in the 5ʹ untranslated region of this mRNA. However, little is known about how Hfq protein contributes to the annealing of other sRNAs to their mRNA targets, especially those, which exert negative regulation of translation and bind to the coding sequence of their mRNA targets. To address this issue the contribution of Hfq to the annealing of three sRNAs to the coding sequence of Salmonella ompD mRNA was explored. The results presented in this thesis showed that Hfq protein interacted with ompD mRNA in direct and specific way and significantly accelerated the rate of RybB and MicC sRNA association with this mRNA, while it had a smaller effect on the SdsR-ompD complex formation. The experiments using truncated, unstructured sRNA and mRNA molecules suggested that the Hfq protein differently contributed to the annealing of each sRNA molecule to ompD mRNA. In vitro structure probing revealed that the ompD mRNA leader sequence folds into 5 stem–loop structures with RybB, SdsR, and MicC binding sites located in different structural contexts, which may explain differences in Hfq contributions to their interactions. Additionally, the binding of RybB and MicC sRNAs induced conformational changes in ompD mRNA leader, which were consistent with local unfolding of mRNA secondary structure. Finally, the results indicated that the binding of Hfq to the long AU-rich sequence located in the 5′-untranslated region of ompD mRNA was essential for the Hfq-dependent annealing of sRNAs to the mRNA coding sequence. Overall, the data showed that Hfq assists small RNAs in binding to the ompD mRNA coding sequence but its specific contributions depend on the sequence and structure of interacting RNA molecules.
Record ID
UAMa66b08cd9c124ea69a4b2a90d17cea60
Diploma type
Doctor of Philosophy
Author
Title in Polish
Rola białka Hfq w oddziaływaniu niekodujących RNA bakterii z sekwencją kodującą mRNA
Title in English
The contribution of the Hfq protein to interactions of bacterial noncoding RNAs with the mRNA coding sequence
Language
pol (pl) Polish
Certifying Unit
Faculty of Biology (SNP/WB/FoB) [Not active]
Discipline
biochemistry / (biological sciences domain) / (biological sciences)
Status
Finished
Year of creation
2016
Start date
18-03-2018
Defense Date
21-06-2016
Title date
24-06-2016
Supervisor
Keywords in English
Hfq, sRNA, coding sequence, ompD, mRNA
Abstract in English
In the presented PhD thesis the contribution of Hfq protein to the annealing of small noncoding RNAs (sRNAs) to the complementary sequences in a target mRNA was analyzed. So far, the detailed molecular mechanism used by Hfq has been described only for its role in the positive regulation of E. coli rpoS mRNA by DsrA sRNA, which recognizes a sequence located in the 5ʹ untranslated region of this mRNA. However, little is known about how Hfq protein contributes to the annealing of other sRNAs to their mRNA targets, especially those, which exert negative regulation of translation and bind to the coding sequence of their mRNA targets. To address this issue the contribution of Hfq to the annealing of three sRNAs to the coding sequence of Salmonella ompD mRNA was explored. The results presented in this thesis showed that Hfq protein interacted with ompD mRNA in direct and specific way and significantly accelerated the rate of RybB and MicC sRNA association with this mRNA, while it had a smaller effect on the SdsR-ompD complex formation. The experiments using truncated, unstructured sRNA and mRNA molecules suggested that the Hfq protein differently contributed to the annealing of each sRNA molecule to ompD mRNA. In vitro structure probing revealed that the ompD mRNA leader sequence folds into 5 stem–loop structures with RybB, SdsR, and MicC binding sites located in different structural contexts, which may explain differences in Hfq contributions to their interactions. Additionally, the binding of RybB and MicC sRNAs induced conformational changes in ompD mRNA leader, which were consistent with local unfolding of mRNA secondary structure. Finally, the results indicated that the binding of Hfq to the long AU-rich sequence located in the 5′-untranslated region of ompD mRNA was essential for the Hfq-dependent annealing of sRNAs to the mRNA coding sequence. Overall, the data showed that Hfq assists small RNAs in binding to the ompD mRNA coding sequence but its specific contributions depend on the sequence and structure of interacting RNA molecules.
Thesis file
  • File: 1
    praca_doktorska_Zuzanna_Wroblewska_2016.pdf
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Uniform Resource Identifier
https://researchportal.amu.edu.pl/info/phd/UAMa66b08cd9c124ea69a4b2a90d17cea60/
URN
urn:amu-prod:UAMa66b08cd9c124ea69a4b2a90d17cea60

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