Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli
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ARTICLE
Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli Andreas Schedlbauer1 · Idoia Iturrioz1 · Borja Ochoa‑Lizarralde1 · Retina Çapuni1 · Xu Han1 · Elisa de Astigarraga1 · Tammo Diercks1 · Paola Fucini1,2 · Sean R. Connell1,2 Received: 18 December 2019 / Accepted: 6 July 2020 © Springer Nature B.V. 2020
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. Keywords NMR · Ribosomal maturation factor · RbfA · Multidimensional NMR spectroscopy 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 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12104-020-09969-0) contains supplementary material, which is available to authorized users. * Paola Fucini [email protected] * Sean R. Connell [email protected] 1
Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Bizkaia, Spain
Basque Foundation for Science, IKERBASQUE, 48011 Bilbao, Spain
2
Biological context During ribosome biogenesis, a number of assembly factor proteins modify the folding landscape of the ribosome by promoting structural conformations that guide proper RNA folding and/or protein-RNA interactions. The ribosome assembly factor RbfA from Escherichia coli (UniProt: P0A7G2; 15,154 Da; 133 residues) binds to the 30S ribosomal subunit
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