Inorganic pyrophosphatase

ATP hydrolysis capacity of CtUba4 WT and mutants in the presence of CtUrm1 protein was measured using the Malachite Green Phosphate Assay Kit (Sigma‐Aldrich). The reactions were performed in a buffer composed of 100 mM MES pH 6.0; 100 mM NaCl; 2 mM MgCl₂; and 160 μM ATP at 37°C for 90 min. Final CtUba4 and CtUrm1 concentrations were 40 and 60 μM, respectively. The reaction was conducted in the presence of inorganic pyrophosphatase (Thermo Fisher Scientific) in order to catalyzes the hydrolysis of inorganic pyrophosphate generated by Uba4 into phosphate molecules. After the incubation, reaction mixtures were diluted 20 times with water and the orthophosphate concentrations were measured according to manufacturer's instructions. The absorbance was measured using a SpectraMax^® 190 Microplate Reader (Molecular Devices). Three independent experiments were performed, each with two technical replicates.

T7::sgRNA expression cassettes were amplified from their respective pJET1.2 host vectors using primers listed in Additional file 1: Table S3. One microgram of gel-purified linear DNA was used as a template in an in vitro transcription reaction using 100 U of T7 RNA Polymerase (EP0111, Thermo Fisher), 0.1 U inorganic pyrophosphatase (EF0221, Thermo Fisher), 40 U RiboLock RNase Inhibitor (EO0381, Thermo Fisher), and 10 mM NTP mix (R0481, Thermo Fisher) in 1× T7 RNA Polymerase buffer for 16 h at 37 °C. Transcribed sgRNAs were purified by phenol - chloroform extraction.

The sgRNA was transcribed from a dsDNA template (Supplementary Table 1) in a 5 ml transcription reaction (30 mM Tris-HCl pH 8.1, 25 mM MgCl2, 2 mM spermidine, 0.01% Triton X-100, 5 mM CTP, 5 mM ATP, 5 mM GTP, 5 mM UTP, 10 mM DTT, 1 µM DNA transcription template, 0.5 units inorganic pyrophosphatase (Thermo Fisher), 250 µg T7 RNA polymerase). The transcription reaction was incubated at 37 °C for 5 h, after which the dsDNA template was degraded for 30 min with 15 units of RQ1 DNAse (Promega). The transcribed sgRNA was PAGE purified on an 8% denaturing polyacrylamide gel containing 7 M urea, ethanol precipitated and dissolved in DEPC-treated water.

A dsDNA construct containing a T7 promoter and a downstream G-core (5′-TT(GGGGA)₃GGGGTT-3′) was prepared by overlap extension polymerase chain reaction. Transcription was carried out with 0.05-μM dsDNA at 37°C for 1 h in 20 μl of transcription buffer of 40-mM Tris-HCl (pH 8.0), 30% (w/v) Dimethyl sulfoxide (DMSO), 50-mM KCl, 2 U/μl of T7 RNA polymerase (Thermo Scientific, MA, USA), 8-mM MgCl, 10-mM Dithiothreitol (DTT), 2-mM spermidine, 2-mM adenosine triphosphate (ATP), Uridine triphosphate (UTP) and Guanosine monophosphate (GMP), 1-mM Guanosine triphosphate (GTP) or 7-deaza-GTP and 0.005 U/μl inorganic pyrophosphatase, (Thermo Scientific, MA, USA). An equal volume of buffer containing 30% DMSO, 50-mM KCl, 1 μM of competitive DNA and the indicated RNases (0.8-μg/μl RNase A, 0.4-U/μl RNase H) was added and the mixture was maintained at 37°C for 1 h to terminate the transcription and digest the RNA. DNA samples were then resolved at 4°C, 8 V/cm on a 10% polyacrylamide gel in 1× Tris-borate-ethylenediaminetetraacetic acid (EDTA) buffer that contained 75-mM KCl (13 (link),19 (link)) and DNA detected by the fluorescence of carboxyfluorescein (FAM) dye labeled at the 5′ end of the non-template strand using a Typhoon 9400 phosphor imager (GE Healthcare, PA, USA).

IPTG, ATP, HEPES and MgCl₂ were purchased from AppliChem. TCEP was purchased from Carl Roth. Desulfo-coenzyme A and AMP were purchased from Jena Bioscience. AF546 maleimide and inorganic pyrophosphatase were purchased from Thermo Fisher Scientific. Ni-NTA agarose was purchased from Cube Biotech GmbH. Oligonucleotides were purchased from Biolegio. Other chemicals were purchased from AppliChem, Roth or Thermo Scientific. Experimental data were analyzed and fitted using GraphPad Prism 7.