RNase E cuts site-specifically near the CCA-(3′)-end of the ssrA RNA. In vitro cleavage assays were performed for 0, 2, 10, 30, and 60 min durations with (+) or without (−) RNase E complexes. ssrA RNA precursors (pSsrA) used in these assays were continuously labeled with [α-³²P]UTP (a), were 5′ labeled with [γ-³²P]ATP (b), or were unlabeled (c). (a and b) The cleavage reactions were separated by gel electrophoresis using an 8% polyacrylamide sequencing gel and were subjected to autoradiography. The positions of ssrA RNA precursors (pSsrA) and RNase E cleavage products (I and II) are shown. (c) 5′-end termini of RNase E cleavage products were detected by primer extension analysis as described (23) using a 5′ labeled 10Sa-5 primer whose sequences are complementary to the sequences of nucleotides 439–450 of ssrA RNA precursor (see Experimental Procedures and d). The DNA sequencing ladders (c, lanes 5–8) of the ssrA gene corresponding to the 5′-end termini detected for the primer extension are shown. The RNase E processing sites of the ssrA RNA precursor near the CCA-(3′)-end of matured ssrA RNA are indicated with arrows (c and d), in which the arrow with a longer bar indicates a dominant cutting site. The ssrA RNA precursor, some of the coordinators of the ssrA RNA precursor, the RNase P, and RNase E processing sites and the DNA primers used for the experiments are shown (d). The region containing the ORF of the carboxyl-terminal tag (Tag) and its stop codons (asterisks) are marked.

Northern blot analysis of ssrA RNA and ssrA RNA precursors before (U lanes) or after (I lanes) induction. The lower panel shows the internal control of ethidium bromide stained 5S rRNA. The positions of ssrA RNA (SsrA), ssrA RNA precursor (pSsrA), and a read-through fusion-transcript of the λ repressor variant mRNA-ssrA RNA precursor (λ-pSsrA) also are shown. The λ-pSsrA fusion transcript was confirmed by Northern blot detection using a ³²P-labeled riboprobe complementary to λ repressor variant mRNA (data not shown). This experiment was carried out in parallel to the experiment of Fig. 3: isopropyl β-d-thiogalactoside (1 mM) was added at an OD₄₆₀ of 0.4 and was divided into two cultures incubated at 30 and 44°C, respectively, for 20 min (see Experimental Procedures, “Pulse–chase experiments”). Immediately before the pulse–chase, 2 OD₄₆₀ units of each culture were removed for total RNA preparation for Northern blot analysis (35). It is expected to detect small amount of stable, matured ssrA RNA, which were made on isopropyl β-d-thiogalactoside induction and prior complete inactivation of RNase E.

RNase E inactivation causes the accumulation of ssrA RNA precursors with an unmatured 3′ end. rne wild-type (wt) or temperature-sensitive (rne-3071 ts) FLAG-RNase E, pRE196, or pRE205 (16), respectively, was overexpressed in the N3431 (rne-3071) carrying pGP1−2 (34) at 44°C for 30 min. Total RNAs (T) or RNAs associated with RNase E complexes (C) were isolated from cell lysates or affinity-purified RNase E complexes as described in Experimental Procedures. (a) The purified RNAs were loaded onto a 5% polyacrylamide sequencing gel and were stained with ethidium bromide (lanes 1–4), and the RNAs were transferred onto a Zeta-Probe blotting membrane (Bio-Rad) and were hybridized with riboprobe complementary to ssrA RNA (lanes 5–8). The positions of 23S, 16S, and 5S rRNAs, mature ssrA RNA (SsrA), and ssrA RNA precursors (pSsrA) are shown. (b) Matured 5′ end termini of ssrA RNAs were detected by primer extension analysis (lanes 1–4) using a synthetic DNA primer 10Sa-1 complementary to the sequences of nucleotides 27–51 of ssrA RNA as indicated in Fig. 2d [10Sa-1, 5′TCG GCA TGC ACC TTG GGT TTC GCA A (22)]. DNA sequencing ladders generated by using the identical primer, i.e., 10Sa-1, are shown (lanes 5–8). The 5′-end sequences of the ssrA RNA precursor (labeled as pSsrA at the −7 nucleotide position) or matured ssrA RNA (shown as 1) and RNase P processing site (labeled with an arrow; also see Fig. 2d) also are shown.

RNase E activity is required for the ssrA RNA mediating carboxyl-terminal tagged proteolysis. (a) Degradation of λ repressor (residues 1–93)-M2-H6-trpAt protein in WCL01 (ssrA⁻ rne-3071) containing pPW500 [for overexpression of λ repressor variant (28)] or pPW510F (for overexpression of λ repressor variant and ssrA RNA precursor) cultured at 30°C (active RNase E) or 44°C (inactive RNase E) assayed by pulse–chase experiments. The λ repressor variant mRNA and ssrA RNA precursors were expressed by isopropyl β-d-thiogalactoside induction as described in Experimental Procedures. The positions of [³⁵S]Met-pulse-labeled newly synthesized polypeptides, the λ repressor variant protein (λ), and the ssrA RNA-mediated peptide tag (λ-tag) are indicated at 0 min (lanes 2, 5, 9, and 12). Lane U, uninduced control. Lanes indicated as 4 and 16 min: time after chase. (b) Degradation of the pulse-labeled λ repressor occurs in the rne wild type containing pPW510F cultured at both 30°C (lanes 2–4) or 44°C (lanes 5–8).

Schematic model showing events in which a partially degraded mRNA is released from 70S ribosomes by ssrA RNA charging of the CCA-3′ terminus generated by RNase E with an alanine that promotes trans translation of an ssrA-encoded ORF specifying peptide tag used in proteolysis. 70S ribosomes (indicated as two joining circles), elongating nascent polypeptides (streams of small circles), broken mRNA lacking a stop codon (curved line), carboxyl terminal ssrA-encoded peptide-tag (filled circles), RNA polymerase complex (RNAP), and DNA template are shown. N indicates the NH₂-terminal of peptides. Ala-charged ssrA RNA/tm RNA is shown.