FIG 7 .

Analysis of VirF₂₁ translation from the virF R2 transcript. (A) Primer extension analysis of total RNA from BL21(DE3) cells carrying pAC-T730FT or pACT7-HH-21-FT with or without induction using IPTG. The arrowhead indicates the position of hammerhead cleavage. (B) Western blot analysis of total protein extracts from strain BL21(DE3) cells carrying pAC-T730FT or pACT7-HH-21-FT, with or without induction by IPTG. Shown is translation of both VirF₃₀ and VirF₂₁ in the presence of pACT7-HH-21-FT or of only VirF₂₁ in the presence of pACT7-HH-21-FT. (C) In vitro translation of virF R1-3XFT (start, + 1) and R2-3XFT (start, + 309) mRNAs. Both VirF₃₀ and VirF₂₁ were translated from virF R1-3XFT, but only VirF₂₁ was translated from virF R2-3XFT mRNA. Asterisk, unspecific cross-hybridization with a protein in the extract. In the blot on the right, we included ompF mRNA as an internal canonical, SD-dependent translation control. (D) Toeprint assay results with +1 (full-length) and +309 (leaderless) virF mRNAs. The mRNAs were incubated alone (control; lanes 1 and 4), with 30S (lanes 2 and 5), or with 30S and tRNA^−fMet (lanes 3 and 6). A specific toeprint was observed on full-length mRNA (+1) near the 5′ end (black circle) (compare with Fig. 2C). A second toeprint, specific to the llmRNA, is at position +326 (black asterisk). Sequencing ladders were generated with the same 5′-end-labeled primer.