Figure 4.

RNase Y-dependent cleavage occurs 6 nt upstream of putative recognition determinant. (A) Schematic representation of constructs pCG392 and pCG394 carrying a deletion downstream of CS and a deletion of both aCS and CS, respectively (deletions are indicated in the panel as a cross). The RNAs detected in the northern blot analysis are shown below each construct. (B) Northern blot analyses to examine sae processing in strains carrying pCG392 and pCG394. Newman wild-type and rny mutant strains carrying the different constructs were grown to exponential phase (Newman pCG212 was included in the analysis as a control). RNA was then harvested and hybridized to DIG-labeled DNA probes specific for gfp and rny. As a loading control, 16S rRNA was detected in the ethidium bromide-stained gel, as shown at the bottom of the panel. The processed RNA found in construct pCG394 is indicated by *. (C) Mapping of the 5΄ end of the processed RNA in construct pCG394. The sae sequence is aligned with those of three different clones (1, 2 and 3). The RNA 5΄ adapter is underlined, and the mapped position for alternative cleavage site 2 (aCS2) is shown in bold. The deletion in construct pCG394 and the shift in the CS are also indicated. (D) The RNase Y recognition sequence (Rrs) is underlined; the distance between Rrs and the cleavage sites (CS, aCS or aCS2) is underlined with dots.