Two forms of SraG RNA. (A) Genomic context of sraG. The gene encoding SraG (shaded in gray) is located between the coding regions of the rpsO and pnp genes (encoding ribosomal protein S15 and PNPase, respectively). The sraG and pnp genes are expressed divergently but the 5′-ends of these transcripts overlap. Promoters P1, P2, and P, as well as terminators T1, T2, and T for rpsO, pnp, and sraG, respectively, are indicated. Note that T1 and T are the same bidirectional terminator. (B) Two forms of SraG. Wild-type N3433 and bacteria lacking SraG (mutation in the −10 sequence of SraG promoter [PSraGmut] identified in this study) were harvested in exponential and stationary phase and RNA extracted and separated on an acrylamide gel. Northern blots were probed to detect SraG RNA using an antisense RNA probe equivalent to positions 43–179 of the full size SraG identified here and to 5S RNA. Size of the SraG transcripts was estimated by comparison with radiolabeled markers. (C) Schematic representation of the processing of the rpsO and pnp primary transcripts. The monocistronic rpsO, pnp (1), and sraG (3) transcripts and the dicistronic rpsO-pnp mRNA (2) are shown with the black circle indicating their 5′-triphosphate extremities corresponding to P1 (rpsO promoter), P2 (pnp promoter), and P (sraG promoter). Processing sites by RNase E (RE) and RNase III (RIII) on the primary transcripts are indicated by gray and black scissors, respectively. Data on SraG are from experiments shown in Supplemental Figure S5A–C. Potential hairpins within the transcripts are shown: T1 and T2 are the rpsO and pnp terminators, and R is the stem–loop in the 5′-region of pnp subject to RNase III cleavage. (D) SraG primary transcript is 216 nt long. The sequence corresponds to the (+) strand (as shown in A) from the beginning of the pnp ORF to the rpsO termination codon. The sequence highlighted in gray corresponds to the SraG primary transcript identified by cRT-PCR. A total of 76 RT-PCR sraG clones were sequenced. Vertical arrows denote extremities located by cRT-PCR. Arrows in boxes 1 and 2 correspond to the positions of 5′-ends and in boxes 3 and 4 to 3′-extremities. In most cases, numerous clones were detected at each single position. The transcription initiation site (underlined T in box 1) was only detected in samples after polyphosphatase treatment (see Supplemental Fig. S2). Both the previously annotated 5′-extremity and the newly described +1 transcription start site are shown in bold and underlined. The −10 and the −35 sequences are underlined. The 3′-extremity of the terminator, which is the major 3′-extremity, is shown in bold and underlined, but a few clones harbor 3′-ends downstream, indicating that some read-through occurs. The 190-nt long RNA should correspond to a population of RNA molecules, with different 5′- and 3′-extremities. While a fraction of these RNA fragments, which starts at PSraG or very close (panel D, box 1), have their 3′-ends in box 3 (176- to 185-nt long fragments), the rest of the shorter SraG transcripts could correspond to a 5′-end in box 2 (panel D) and a stop at the terminator (174- to 189-nt long fragments) (panel D, box 4). These RNA fragments of ∼190 nt are hereafter collectively designated as SraGp. Boxes TTG and TAA in gray indicate the pnp START codon and rpsO STOP codon located on the (−) strand, respectively. The pnp transcription initiation site and the pnp ribosome-binding site (RBS) located on the (−) strand are also indicated in gray.
