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. 2000 Dec 1;28(23):4623–4633. doi: 10.1093/nar/28.23.4623

Figure 7.

Figure 7

RNA processing of 23S rRNA precursors of Bradyrhizobia and R.sphaeroides in vitro using RNase III purified from E.coli or R.sphaeroides. (A) Structure of DNA region and transcripts derived from plasmids bearing cloned PCR fragments from 23S rRNA genes of various α-proteobacteria. Upper portion shows the structure of a representative template containing an internal segment from 23S rRNA which bears the 5′ IVS. PCR fragments were made with primers complementary to conserved regions of 23S rRNA and are represented by the broad black line. DNA fragments derived from PCR synthesis on R.palustris, Bradyrhizobium USDA 4362, R.sphaeroides, Bradyrhizobium USDA 4377 and B.japonicum genomic DNA templates were cloned in the pGEM-T vector. Plasmid DNAs were cleaved at the ScaI site found in all of these 23S rRNA genes and transcribed in vitro from the T7 promoter on the vector portion (T7P) using T7 RNA polymerase. Lower line shows the proposed in vitro processing pathway of the IVS-containing precursor in those cases where RNase III cleavage occurs. Non-concerted cleavage by RNase III at sites indicated by the arrows in stem–loop #1 produces RNA fragments from the left side (L), right side (R) and the IVS (I). Intermediates (L+I) and (R+I) are produced in unequal amounts in each case due to variable cleavage on either side of stem–loop #1. (B) Ethidium bromide-stained 6.25% acrylamide gel showing electrophoretic separation of the products of processing reactions carried out for 0 h (lanes 2, 4 and 6) and 2 h (lanes 3, 5 and 7) in the presence of 0.5 µl his-tagged E.coli RNase III. Lane 1, RNA marker (Gibco-BRL); lanes 2–3, R.palustris 23S rRNA precursor; lanes 4–5, Bradyrhizobium USDA 4362 rRNA precursor; lanes 6–7, R.sphaeroides 23S rRNA precursor. (C) Ethidium bromide-stained 7.5% acrylamide gel showing electrophoretic separation of the products of processing reactions carried out for 0 h (lanes 2 and 4) and 2 h (lanes 3 and 5) in the presence of 0.5 µl his-tagged E.coli RNase III. Lane 1, as above; lanes 2–3, Bradyrhizobium USDA 4377 rRNA precursor; lanes 4–5, B.japonicum 23S rRNA precursor. (D) Ethidium bromide-stained 7.5% acrylamide gel showing electrophoretic separation of the products of processing reactions carried out for 0 min (lanes 2 and 6), 10 min (lanes 3 and 7) and 30 min (lanes 4 and 8) in the presence of 1.0 µl partially purified R.sphaeroides RNase III or 30 min with 1.0 µl his-tagged E.coli RNase III (lanes 5 and 9). In (B), (C) and (D), RNA marker sizes are indicated on the left margin of the gel and completely processed products are indicated on the right margin (R and L). These are derived by cleavage as shown in (A). P, position of the rRNA precursor in each case; I [in white within (B), (C) and (D)], IVS-containing cleavage products from USDA 4362, USDA 4377 B.japonicum and R.palustris from left to right, respectively. Products proposed to be processing intermediates resulting from non-concerted RNase III cleavage (L+I and R+I) based on their size, run at positions between bands L and R and between R and P, respectively, and are marked by white arrowheads on the side of lanes 3 and 5 in (B) and (C) and lane 5 in (D).