Figure 3
The effect of gene disruption on Ψ formation in the domain V of T. kodakarensis 23S rRNA. (A) Ψs identified in this work in the T. kodakarensis 23S rRNA, which are conserved in the P. abyssi 23S rRNA are indicated by black dots. The grey dots correspond to other Ψs detected in P. abyssi 23S rRNA in a previous work55. The secondary structure of the T. kodakarensis 23S rRNA is adapted from Piekna et al.16. The sequence of the two substrates mini–23S–2607 and mini–23S–2589, synthesized by in vitro transcription and used in vitro in Panel C (mini–23S–2607) and in Fig. 5 (mini–23S–2589), is highlighted. (B) Identification of Ψs in the T. kodakarensis 23S rRNA by the CMCT-RT method. As indicated at the top of each lane, total RNAs extracted from the various T. kodakarensis strains were treated in the absence (−) or the presence of CMCT (+), for 2, 10, and 20 min as described in Methods. The CMCT treatment was (+) or was not (−) followed by an alkaline treatment at pH 10.4. The positions of Ψs were identified by primer extension analysis using oligonucleotide O-2941 (represented by the dashed arrow) as described in Methods. Digital image of the autoradiogram was obtained by scanning the x-ray film. The presence of Ψs is revealed by the appearance of alkali-resistant RT stops. Lanes U, G, C, and A correspond to the RNA sequencing ladder. The positions of nucleotides in 23S rRNAs are indicated to the left of the autoradiogram. The two panels correspond to a cropping of two sections of the same autoradiogram. The full-length gel is presented in Supplementary Figure S4. (C) Time course analysis of in vitro modification by recombinant proteins in RNA substrate mini–23S–2607. The RNA substrate was radiolabeled by the incorporation of [α-32P]CTP during in vitro transcription. The RNA was incubated with different protein combinations: Cbf5 alone (C), Cbf5 and Nop10 (CN), Cbf5, and Gar1 (CG), and a set of the three proteins (CNG). At each time point, an aliquot of the reaction mixture was analyzed by 2D-TLC. The radioactivity was quantified by PhosphorImager analysis. The quantities of Ψ nucleotides formed are expressed in moles per mole of mini–23S–2607.
