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. 2014 Dec 23;12(12):e1002028. doi: 10.1371/journal.pbio.1002028

Figure 7. Biochemical analysis of CYT-18/Sc mtTyrRS chimeric proteins.

Figure 7

Chimera 1 consists of the CYT-18 NTDs fused to the Sc mtTyrRS linker region and CTD, while chimera 2 consists of the CYT-18 NTDs and linker region (including Ins 3) fused to Sc CTD. Tyrosyl-adenylation, aminoacylation, and RNA-splicing assays were done at 30°C, as described in Materials and Methods. (A) Tyrosyl-adenylation activity is displayed as a bar graph showing the mean for three experiments, with the error bars indicating the standard deviation. (B) Aminoacylation assay showing the formation of [3H]-Tyr-tRNATyr over a 60-min time course (black open circles, wild-type CYT-18; purple open squares, chimera 1; pink open diamonds, chimera 2; black closed circles, no protein). (C, D) End-point splicing assays of 32P-labeled precursor RNAs (200 nM) containing the Nc mt LSU and ND1m group I introns, respectively, with 100 nM protein and 1 mM GTP for 60 min at 30°C in splicing reaction medium containing 25 or 100 mM KCl. Additional end-point splicing assays with 32P-labeled precursor RNA at these protein and RNA concentrations at 25°C and 37°C are shown in Figure S7A and S7B. (E, F) Splicing time courses of 32P-labeled precursor RNA containing the ND1m intron (200 nM) with 100 nM protein and 1 nM GTP at 30°C in splicing reaction medium containing 25 mM or 100 mM KCl, respectively. The plots show disappearance of precursor RNA as a function of time (black open circles, wild-type CYT-18; purple open squares, chimera 1; pink open diamonds, chimera 2). (G) End-point splicing assays with unlabeled precursor RNA containing 200 nM Nc mt LSU intron and [α-32P]GTP (500 nM; 3,000 Ci mmol−1) with 500 nM protein. Reactions were incubated for 60 min at 30°C and 37°C. Darker exposures of the same gels are shown below. Minor labeled products in the CYT-18 lanes, including one co-migrating with precursor RNA and others migrating above precursor RNA (not shown), appear in time-course experiments after the 5′-labeled intron products and likely reflect secondary reactions catalyzed by group I intron RNAs (see [87]). The left panel shows splicing reactions for the same concentrations of wild-type CYT-18 protein and 32P-labeled precursor RNA at 30°C run in parallel as a control. Abbreviations: E1-E2, ligated exons; E1-I, 5′ exon+intron; I, excised intron; I-E2, intron+3′ exon; P, precursor RNA.