Aregger, Regula Helena. The impact of the linker in the catalytic core of the DEAD-box protein YxiN from Bacillus subtilis on substrate binding, enzymatic function and conformation. 2012, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10205
YxiN is a DEAD-box RNA protein from Bacillus subtilis that recognises hairpin 92 of the 23S rRNA. The protein consists of a catalytic core formed by two RecA domains connected by a linker of 9 amino acids and a C-terminal domain conferring substrate specificity. The sequence motifs for ATP binding, ATP hydrolysis and RNA binding are located in the two RecA domains of the helicase catalytic core and communication between these two domains is crucial for enzymatic function.
To investigate the role of the linker between the two RecA domains of the DEAD-box helicase core, its length and sequence were modified. RNA stimulated ATPase activity of YxiN mutants and YxiN wildtype was characterized by a coupled enzymatic steady-state ATPase assay and unwinding assays were performed using a double stranded RNA minimal substrate. Nucleotide binding affinities were determined by displacement titrations of nucleotide with a preformed complex of enzyme with a fluorescently modified nucleotide in the absence and the presence of a minimal RNA substrate. RNA binding affinities were determined by anisotropy titrations of a fluorescently labeled minimal substrate with YxiN wildtype and YxiN mutants. Conformational changes were investigated in confocal smFRET experiments.
This work could show that extension of the linker up to 15 amino acids led to a drastic reduction of ATPase activity. This effect was even more pronounced in mutants with shortened linkers. Reducing the linker length to 4 amino acids resulted in a completely ATPase-deficient enzyme. However nucleotide binding studies of the different mutants showed that ATP affinity is increased in all the mutants when compared to the wildtype enzyme. If additionally an RNA substrate is present the increase in ATP affinity thus can only be observed for the shorter YxiN linker mutants and the longer YxiN linker mutants rather show a decrease in ATP affinity. The affinities for the pre-hydrolysis ATP-analogue ADP•BeFx are lower for the YxiN linker mutants compared to YxiN wildtype in the absence of RNA, but similar to YxiN wildtype in the presence of RNA. In the absence of RNA the affinities of the YxiN linker mutants for ADP are similar to those found for YxiN wildtype but are clearly decreased in the presence of RNA. The linker in the catalytic core also has some influence on RNA binding as reflected by the increase in affinity for RNA in the YxiN linker mutants compared to YxiN wildtype in the absence of nucleotide and in the presence of ADP•BeFx. Changing the linker length leads to lower RNA affinities in the presence of ADPNP and to higher RNA affinities in the presence of ADP. The same trends seen for ATPase activity are observed for unwinding activities of these mutants with the longest and shortest constructs being unwinding deficient. Differences in unwinding rates depending on the nucleotide present could be observed. Unwinding rates with ATP are in general higher than unwinding rates with ADP•BeFx. Single molecule FRET experiments indicated that mutants with the shortest and the longest linkers did not adopt a closed conformation in the presence of RNA substrate and ATP but in all other YxiN mutants as well as in YxiN wildtype.
These results indicate that the linker between the catalytic core RecA domains of YxiN plays a key role in regulation of the enzyme activity. Furthermore, it is evident that the length of the linker for efficient interdomain communication and thus enzymatic activity between the two core domains lies between 5 and 13 amino acids.
|Committee Members:||Seelig, Joachim|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Klostermeier)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||126 Bl.|
|Last Modified:||30 Jun 2016 10:51|
|Deposited On:||09 Jan 2013 14:12|
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