Abstract
RbfA (ribosome binding factor A; 15.2 kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15 N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293 K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.
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Abbreviations
- IPTG:
-
Isopropyl-thio-β-D-galactoside
- E. coli :
-
Escherichia coli
- RbfA:
-
Ribosome binding factor A
- PIC:
-
Protease inhibitor cocktail
- TCEP:
-
Tris(2-carboxyethyl)-phosphine
- RCI:
-
Random coil index
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Acknowledgements
This work was supported by grants from the European Union’s Seventh Framework Program (Marie Curie Actions; COFUND; to S.R.C. and A.S.), Marie Curie Action Career Integration Grant (PCIG14-GA-2013-632072 to P.F.), and Ministerio de Economía y Competitividad Grant (CTQ2017-82222-R to P.F., S.R.C.). We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV-2016-0644) and the proteomics platform of CIC biogune for sample analysis by MALDI TOF mass spectroscopy.
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Schedlbauer, A., Iturrioz, I., Ochoa-Lizarralde, B. et al. Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli. Biomol NMR Assign 14, 317–321 (2020). https://doi.org/10.1007/s12104-020-09969-0
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DOI: https://doi.org/10.1007/s12104-020-09969-0