Uncovering the Stoichiometry of <i>Pyrococcus furiosus</i> RNase P, a Multi‐Subunit Catalytic Ribonucleoprotein Complex, by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

Tanimoto

et al. 2017

Nucleic Acids Research

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RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented ‘double K-turn’ module in type A and type M archaeal RPR variants.

“…RPRs were refolded as described earlier for MS (35), footprinting (32) and pre-tRNA processing (11,16) experiments. See Supplementary Information for additional details.…”

Section: Methodsmentioning

confidence: 99%

A novel double kink-turn module in euryarchaeal RNase P RNAs

Tanimoto

et al. 2017

Nucleic Acids Research

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“…138–144 This method has provided an exceptional means to reconstruct the quaternary structures of protein complexes, as initially demonstrated for transthyretin tetramers and serum amyloid P decamers, 137 then subsequently reported for complexes comprised of tetradecameric GroEL, 138 dimeric phosphorylase B, 140 and hexameric glutamate dehydrogenase, 140 as well as a multi-unit ribonucleoprotein complex. 141 Examples of the SID and CID mass spectra obtained for tetrameric complexes of streptavidin, neutravidin, and transthyretin are shown in Figure 6. 142 The SID spectra at various collision energies reveal that these tetrameric proteins dissociate to yield monomeric and trimeric species, as well as dimeric ions not observed upon CID.…”

Section: Native Proteins and Protein Complexesmentioning

confidence: 99%

Ion Activation Methods for Peptides and Proteins

2015

“…In this study nanoelectrospray ionization mass spectrometry (nano-ESI MS) was used to determine the AmrZ oligomeric state in solution. Mass spectrometry precisely measures the size of a molecule and nano-ESI MS allows the detection of proteins or protein complexes when they are still folded (Karas et al, 2000;Ma et al, 2014). As shown in Fig.…”

Section: Amrz Forms Tetramers In Solution Via Its Ctdmentioning

confidence: 99%

The Pseudomonas aeruginosa AmrZ C‐terminal domain mediates tetramerization and is required for its activator and repressor functions

Soukup

et al. 2015

Environ Microbiol Rep

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Summary Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher-order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets.