Figure 3.

RNase Y-dependent cleavage is independent from saeP and CS sequences. (A) Schematic representation of construct pCG301 carrying just the region of sae between the two stem loops under the native promoter P1. Below the construct, the RNAs observed in the northern blot analysis are indicated. (B) Northern blot analyses to examine sae processing of the pCG301 construct. Newman wild-type and rny mutant strains carrying pCG301 were grown to the exponential phase, and RNA was harvested and hybridized to a DIG-labeled DNA probe specific for gfp and rny. The 16S rRNA detected in ethidium bromide-stained gel served as a loading control and is shown at the bottom of the panel. (C) Northern blot analyses to examine sae processing in strains carrying the sae-gfp constructs shown in Figure 1B. Newman wild-type and rny mutant strains carrying the different constructs were grown to exponential phase. RNA was then harvested and hybridized to DIG-labeled DNA probes specific for gfp, saeP and rny. As a loading control, 16S rRNA was detected in ethidium bromide-stained gel, as shown at the bottom of the panel. (D) Mapping of the 5΄ end of the processed RNA in construct pCG213 by RACE analyses. cDNA was obtained using RNA from the Newman wild-type strain carrying pCG213. The sae sequence shown in the panel is aligned with those of three different clones (1, 2 and 3). The RNA 5΄ adapter is underlined, and the mapped position for the alternative cleavage site (aCS) is indicated in bold. The deletion in construct pCG213 and the shift of the CS are also shown.