title: Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry.
abstract: The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.
authors.affiliation: Department of Biological Sciences, University of Waikato, Hamilton 3240, New Zealand.
authors.forename: Joanna L
authors.initials: JL
authors.lastname: McKenzie
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Duyvestyn
authors.forename: Tony
authors.initials: T
authors.lastname: Smith
authors.forename: Katerina
authors.initials: K
authors.lastname: Bendak
authors.forename: Joel
authors.initials: J
authors.lastname: Mackay
authors.forename: Ray
authors.initials: R
authors.lastname: Cursons
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
authors.forename: Vickery L
authors.initials: VL
authors.lastname: Arcus
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D041543:Pyrobaculum / Q000201:enzymology; D015347:RNA Probes / Q000737:chemistry*; D012260:Ribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D015347:RNA Probes; D012260:Ribonucleases