Domain orientation in the RNA helicase YxiN and the role of conformational changes for RNA unwinding

Karow, Anne R.. Domain orientation in the RNA helicase YxiN and the role of conformational changes for RNA unwinding. 2010, PhD Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_9216


The RNA helicase YxiN from Bacillus subtilis is a member of the family of DEAD box proteins. YxiN is able to unwind RNA double strands in an ATP-dependent manner. The ability to catalyse RNA rearrangement is in vivo presumably necessary for the bacterial ribosome biogenesis.
YxiN comprises a two-domain helicase core region and a C-terminal RNA binding domain. While crystal structures of the C-terminal core domain and the RNA binding domain separately have been determined before, the structure of full-length YxiN is not known. In the current project the orientation of these three domains to each other was determined employing fluorescence resonance energy transfer (FRET) experiments at the single-molecule level. Therefore the approximate architecture of the full-length enzyme in solution can now be described. The two core domains exhibit a conformation similar to the crystal structure of the DEAD box protein MjDeaD. The RNA binding domain is adjacent to the C-terminal core domain. Presumably the central -sheet of the RNA binding domain faces towards a patch of the core domain that is formed by loops.
During catalysis YxiN undergoes a conformational change. The conformation of the core domains mentioned above is adopted in the absence of substrates and in the presence of RNA, ADP, ATP or ADPNP. In the presence of both RNA and ATP (or ADPNP) the core domains approach each other constituting a closed conformation. During the catalytic cycle this conformational change takes place initially after binding of RNA and ATP. The conformational change is necessary for RNA unwinding. But it is not sufficient since the YxiN_K52Q mutant adopts the closed conformation upon binding of RNA and ATP (or ADPNP) but is unwinding deficient.
Transitions between the open and the closed conformation could only rarely been detected in the FRET experiments on a confocal microscope due to a limited observation time. To be able to monitor the conformation of YxiN on longer timescale the protein was engineered for FRET experiments on a total internal reflection microscope. A protocol was developed that comprises fluorophore double labelling of YxiN and the attachment of a biotin at the protein’s C-terminus. The biotinylation procedure is based on the reaction type of expressed protein ligation. The labelled and biotinylated YxiN construct could be specifically immobilized on a streptavidin coated surface for total internal reflection microscopy. Subsequently, YxiN could be monitored for up to a few seconds.
Expressed protein ligation was furthermore employed to develop a specific fluorophore double labelling strategy for FRET experiments. Employing this strategy a YxiN construct could be generated that carries one certain fluorophore exclusively at one position in the protein. A different fluorophore can attach to the same position or to one further site within the protein. The procedure was therefore termed semi-site-specific double labelling. In comparison with statistic labelling procedures the semi-site-specific double labelling allows for decreasing the sample heterogeneity in FRET experiments.
Taken together, this study revealed the conformation of the three-domain RNA helicase YxiN, its conformational change during catalysis which is essential for the activity of the helicase and the study established protein preparation techniques that provide the basis for further studies on the helicase mechanism.
Advisors:Klostermeier, Dagmar
Committee Members:Grzesiek, Stephan
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Klostermeier)
Item Type:Thesis
Thesis no:9216
Bibsysno:Link to catalogue
Number of Pages:122 S.
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:21 Jan 2011 11:57

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