Fig. 1.

GadY RNA-directed mRNA cleavage. (a) Diagram of the gadXWY region. The gadY promoter mutation is indicated by the X. (b) Levels of the gadX (∼1.0 kb) and gadW (∼1.1 kb) mRNAs in cells without a plasmid, carrying the vector control (pRI), or overexpressing GadY (pRI-GadY). The top band in both panels of (b) is likely to be due to cross hybridization with the 16S rRNA. (c) Diagram of the cat replacement of gadX. (d) Levels of the cat (∼0.8 kb) mRNA in cells carrying the vector control (pRI) or overexpressing GadY (pRI-GadY) in a background (GSO129) in which the gadX promoter and coding sequence were replaced with the cat promoter and coding sequence. (e) Diagram of the cat replacement of gadX and gfp replacement of gadW. (f) Levels of the cat (∼0.8 kb, ∼1.7 kb) and gfp (∼ 0.9 kb, ∼1.7 kb) mRNAs in cells carrying the vector control (pRI) or overexpressing GadY (pRI-GadY) in a background (GSO403) in which the gadX promoter and coding sequence were replaced with the cat promoter and coding sequence and a promoterless gfp gene was inserted downstream and separated from cat by the sequences complementary to GadY. In all cases, total RNA (5 μg each) isolated from cultures grown in LB media to OD₆₀₀ = 0.7 was separated in a 1% agarose−0.05 M MOPS−1 mM EDTA gel and then transferred to a nylon membrane. The individual mRNAs were detected by specific oligonucleotides (gadX-R, gadW-A2, cat-A1 and gfp-R).