Figure 7

Differential mRNA translation between wildtype and rancRNA_s194 deletion strains in xylose medium. (A) Cellular protein extracts (S30) were prepared from H. volcanii wildtype (wt) and rancRNA_s194 knock-out (Δ) cells inoculated in xylose medium for the same time period. Proteins were separated on an 11% SDS-tricine gel and stained with coomassie. The arrow indicates the position of a pronounced protein band seen in the knock-out strain. The marker lane and the two experimental lanes were cropped from different parts of the same SDS gel. (B) Two most strongly upregulated proteins in the rancRNA_s194 knock-out strain compared to wildtype H. volcanii as identified by mass spectrometry. (C) Northern blot analysis of the carbon starvation protein cstA mRNA and mRNA coding for ribosomal protein L10. mRNAs were isolated from the polysomal fraction of sucrose gradients of the wt or the ΔrancRNA_s194 H. volcanii cells grown in xylose-containing medium for the same time. Ethidium bromide-stained 23S rRNA and 16S rRNA (lower gel) served as loading controls. Full-length blots are presented in Supplementary Fig. 9. (D) The distribution of the cstA mRNA between different polysome gradient fractions was monitored by northern blot analysis and compared between wildtype and rancRNA_s194 knock-out H. volcanii cells. In all gradient fractions corresponding volumes were loaded, except for the light gradient fraction (free), where only 50% of the corresponding volume fraction was applied. The asterisk indicates unspecific annealing of the northern blot probe (see Supplementary Fig. 10 for uncropped blots and the ethidium bromide stained gel). (E) Binding of radiolabeled mRNAs (CstA; sugar ABC transporter ATP binding protein, ABC; 8-codon-stop mRNA, 8 c.s.; r-protein L12, L12) to H. volcanii ribosomes was measured by filter binding in the absence (−) or presence of unlabeled competitor rancRNA_s194, or the scrambled version thereof (scr). The mean and standard deviation of three to six independent filter binding experiments is shown. Signals measured in the absence of ribosomal particles were subtracted from all experimental points. The binding data with r-protein L12 mRNA have been performed twice and are identical to the ones shown in Supplementary Fig. 4B. Significant differences were determined using the 2-tailed paired Student’s t-test (***p < 0.001, n.s., not significant). Phosphorimager screens of representative filter binding competition experiments are shown.