Skip to main content
. 2017 Oct 3;114(42):11121–11126. doi: 10.1073/pnas.1707862114

Fig. 3.

Fig. 3.

Complementation of E. coli and yeast RNase P by Aq_880. (A) A. aeolicus Aq_880 is able to functionally replace bacterial RNase P in vivo. E. coli BW cells transformed with expression plasmids for Aq_880 or its variants D138A, D142A, D144A, and D160A were grown at 37 °C under permissive (in the presence of arabinose; +ara) or nonpermissive (in the presence of glucose; +glu) conditions. Already after 1 d, cell growth was detectable in E. coli BW cells containing the expression vector for Aq_880; whereas no complementation was observed with the aspartate to alanine mutants D138A, D142A, and D160A, weak colony formation was seen with D144A. rnpB: E. coli RNase P as positive control. (−), the empty vector as negative control. (B) Expression and solubility of Aq_880 variants in E. coli BW. Soluble (s) and insoluble (is) protein fractions from E. coli BW expressing the Aq_880 variants were analyzed by Western blotting using a polyclonal antiserum to Aq_880. No signal was obtained when E. coli BW was transformed with the empty vector (−) or E. coli rnpB (+), respectively; molecular mass marker (in kilodaltons) indicated on the left. (C) Aq_880 and its variant D144A rescue growth upon deletion of the yeast nuclear RNase P RNA gene RPR1. Two colonies each obtained through rescue by Aq_880 or its variant D144A were applied as spots in 10-fold serial dilution to YPD plates, and the growth of the strains was monitored in parallel to a control (rescue by RPR1). Note the later appearance and smaller colony size of Aq_880 and D144A strains indicating slow growth. (D) Genotyping of Aq_880 and D144A colonies derived from plasmid shuffle. The analysis of a control (rpr1Δ::kanMX4 [RPR1]) and two complementation isolates each (Aq_880, genotype rpr1Δ::kanMX4 [Aq_880]; D144A, genotype rpr1Δ::kanMX4 [Aq_880D144A]) is shown. The deletion of RPR1, the integrity of the chromosomal gene disruption, and the presence of Aq_880 were verified by PCR. The part of the gene interrogated by the genotyping PCR is indicated by a black bar on top of the gene cartoon (RPR1, blue; kanMX4, yellow; Aq_880, magenta; promoter/terminator regions, gray) to the right of each agarose gel panel.