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The mechanism of the action of the chaperone protein Hfq in the pairing of regulatory RNAs MgrR and ChiX with mRNA molecules in Escherichia coli

Joanna Kwiatkowska

Abstract

In bacteria, small noncoding RNAs (sRNAs) are involved in response to changes in environmental conditions. The Hfq protein facilitates the base-pairing of sRNAs to their target mRNAs in Escherichia coli and related bacteria. This protein utilizes three distinct RNA binding sites to facilitate RNA interactions. Based on the sRNAs accumulation profile in cells carrying the Hfq protein with mutations in RNA binding sites, sRNAs were previously divided into two classes. Most tested sRNAs showed an overall decrease in accumulation in the rim mutants. These so-called Class I sRNAs bind to the proximal face of Hfq using their 3ʹ-terminal uridine tails, while their internal UA-rich sequences contact the rim of Hfq ring. mRNAs regulated by these sRNAs bind to the distal face of Hfq using repeated ARN sequences. However, a subset of sRNAs, called Class II, were reduced in the distal and proximal face mutants but the levels of a subset of sRNAs increased in the rim mutants. These results suggested that Class II sRNAs interact with opposite sites of the ring, while their target mRNAs interact with the rim of Hfq. In the presented PhD dissertation, the contribution of Hfq protein to association of Class II sRNAs MgrR and ChiX to eptB or ygdQ and chiP mRNAs, respectively, was studied. Firstly, the secondary structures of MgrR and its regulated mRNA fragments were established. Additionally, the affinity of MgrR to RNA recognition sites in Hfq and its properties in the competition to access to the protein were assessed. Then, KinTek Explorer software was used to globally analyze the kinetics data of MgrR and ChiX annealing to regulated mRNAs in the presence of Hfq and its variants with mutations in RNA binding sites. Finally, the effect of an sRNA competitor on the release of the ChiX-chiP complex from the Hfq protein was tested. Overall, the presented results explained how MgrR is recognized by Hfq. Moreover, the sequence motifs in MgrR, eptB and ygdQ responsible for the binding to the chaperone were indicated. The analysis of kinetics of MgrR and ChiX to regulated mRNAs revealed that mutation in the rim of Hfq was the most detrimental as it trapped each sRNA on the Hfq protein and prevented their annealing to regulated mRNAs. On the other hand, the mutations in the proximal and distal face of Hfq were more detrimental for MgrR than ChiX pairing to respective mRNAs, which suggested that ChiX bound tighter than MgrR to each face of Hfq, which would enable it to remain bound to this protein even when one of the binding sites was mutated. Further studies of the effect of sRNA competitor on ChiX annealing to chiP mRNA in presence of Hfq confirmed the role of the Hfq rim site for chiP mRNA binding to Hfq. Additionally, the analysis of kinetically preferred pathways revealed that the distal face of Hfq is a place for RNA competitor binding, enabling the dissociation of sRNA Class II-mRNA complex from the protein. The effect was confirmed by direct dissociation of ChiX-chiP complex from Hfq induced by sRNA competitor. In summary, the data indicate that besides the major similarities in the modes of both Class II sRNAs binding to Hfq there are also subtle differences between them, which can affect the tuning of sRNA-dependent regulation in the adaptation of bacterial cells to changing environmental conditions.
Record ID
UAMee364d7bbc3347d5afd92a20d610c318
Diploma type
Doctor of Philosophy
Author
Title in Polish
Mechanizm działania białka opiekuńczego Hfq w tworzeniu kompleksów pomiędzy regulatorowymi RNA MgrR i ChiX a cząsteczkami mRNA u bakterii Escherichia coli
Title in English
The mechanism of the action of the chaperone protein Hfq in the pairing of regulatory RNAs MgrR and ChiX with mRNA molecules in Escherichia coli
Language
pol (pl) Polish
Certifying Unit
Wydział Biologii [nowa struktura organizacyjna] (SNP/WB)
Scientific discipline (2.0)
6.4 biological sciences
Status
Finished
Year of creation
2021
Defense Date
27-10-2021
Title date
26-11-2021
Supervisor
Pages
157
URL
https://hdl.handle.net/10593/26438 Opening in a new tab
Keywords in English
Hfq; MgrR; ChiX; Class II sRNA
Abstract in English
In bacteria, small noncoding RNAs (sRNAs) are involved in response to changes in environmental conditions. The Hfq protein facilitates the base-pairing of sRNAs to their target mRNAs in Escherichia coli and related bacteria. This protein utilizes three distinct RNA binding sites to facilitate RNA interactions. Based on the sRNAs accumulation profile in cells carrying the Hfq protein with mutations in RNA binding sites, sRNAs were previously divided into two classes. Most tested sRNAs showed an overall decrease in accumulation in the rim mutants. These so-called Class I sRNAs bind to the proximal face of Hfq using their 3ʹ-terminal uridine tails, while their internal UA-rich sequences contact the rim of Hfq ring. mRNAs regulated by these sRNAs bind to the distal face of Hfq using repeated ARN sequences. However, a subset of sRNAs, called Class II, were reduced in the distal and proximal face mutants but the levels of a subset of sRNAs increased in the rim mutants. These results suggested that Class II sRNAs interact with opposite sites of the ring, while their target mRNAs interact with the rim of Hfq. In the presented PhD dissertation, the contribution of Hfq protein to association of Class II sRNAs MgrR and ChiX to eptB or ygdQ and chiP mRNAs, respectively, was studied. Firstly, the secondary structures of MgrR and its regulated mRNA fragments were established. Additionally, the affinity of MgrR to RNA recognition sites in Hfq and its properties in the competition to access to the protein were assessed. Then, KinTek Explorer software was used to globally analyze the kinetics data of MgrR and ChiX annealing to regulated mRNAs in the presence of Hfq and its variants with mutations in RNA binding sites. Finally, the effect of an sRNA competitor on the release of the ChiX-chiP complex from the Hfq protein was tested. Overall, the presented results explained how MgrR is recognized by Hfq. Moreover, the sequence motifs in MgrR, eptB and ygdQ responsible for the binding to the chaperone were indicated. The analysis of kinetics of MgrR and ChiX to regulated mRNAs revealed that mutation in the rim of Hfq was the most detrimental as it trapped each sRNA on the Hfq protein and prevented their annealing to regulated mRNAs. On the other hand, the mutations in the proximal and distal face of Hfq were more detrimental for MgrR than ChiX pairing to respective mRNAs, which suggested that ChiX bound tighter than MgrR to each face of Hfq, which would enable it to remain bound to this protein even when one of the binding sites was mutated. Further studies of the effect of sRNA competitor on ChiX annealing to chiP mRNA in presence of Hfq confirmed the role of the Hfq rim site for chiP mRNA binding to Hfq. Additionally, the analysis of kinetically preferred pathways revealed that the distal face of Hfq is a place for RNA competitor binding, enabling the dissociation of sRNA Class II-mRNA complex from the protein. The effect was confirmed by direct dissociation of ChiX-chiP complex from Hfq induced by sRNA competitor. In summary, the data indicate that besides the major similarities in the modes of both Class II sRNAs binding to Hfq there are also subtle differences between them, which can affect the tuning of sRNA-dependent regulation in the adaptation of bacterial cells to changing environmental conditions.

Uniform Resource Identifier
https://researchportal.amu.edu.pl/info/phd/UAMee364d7bbc3347d5afd92a20d610c318/
URN
urn:amu-prod:UAMee364d7bbc3347d5afd92a20d610c318

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