FIG. 7.

(A) Northern blot analysis of ywcH transcription. RNA was isolated from cells before (37°C) and after (50°C) heat shock or during the exponential (OD₆₀₀ = 0.2) and stationary (OD₆₀₀ = 1.4) growth phases. Equal amounts of RNA were used for the analysis. The positions of the 23S and 16S rRNAs are indicated. Two bands specifically hybridizing to a ywcH probe were obtained. (B) Mapping of the ywcH 5′ end. RNA was isolated from B. subtilis 168 grown at the indicated temperatures. The sequence ladder used as a size marker was obtained by using the same primer as that used for the primer extension reaction. The putative start codon of translation is indicated (TAC). Whereas the 5′ end of the RNA is upstream from the putative translational start codon in heat-shocked cells, it is downstream from this codon in unstressed cells (arrows at left). (C) S1 nuclease mapping of the 3′ end of nfrA. RNA was isolated from cells before (37°C) and after (50°C) heat shock or from stationary-phase cells grown in NB or NB with glucose and glutamate (GG). Equal amounts of RNA were used for the analysis. Restriction fragments of known lengths and a sequence ladder of a known sequence were used as size markers. Four different signals were obtained in the S1 nuclease reaction. The 912-base fragment was derived from an unprocessed probe. The 875-base fragment (large filled arrow) was obtained by S1 nuclease processing of a probe hybridized to an unprocessed nfrA-ywcH transcript. The 397-base fragment (hatched arrow) was processed at the 5′ end of the stem-loop structure. The 378-base fragment (empty arrow) was processed at the single mismatch within the stem-loop structure. (D) Graphic illustration of the results obtained by S1 nuclease mapping and primer extension. The labeling of the arrows is like that in panel C. The 875-base fragment is not due to RNA processing but is specific for the strategy of the experiment. It indicates that readthrough occurs.