Schematic representation of the rne⁺, rneΔ610, and rneΔ645 alleles. The rneΔ610 mutation encodes a truncated RNase E protein encompassing the first 427 amino acids of the N terminus plus the last 25 amino acids of the C terminus (Ow et al. 2000). The rneΔ645 revertant contains a C → A transversion on serine 418 (TCG → TAG), creating a premature stop codon to generate an RNase E polypeptide with only the first 417 amino acids of the N terminus. The various domains of the wild-type RNase E protein are as described by Vanzo et al. (1998). The figure is not drawn to scale.

Western analysis of various RNase E proteins. Equivalent amounts of total protein lysates (50 μg) were electrophoresed in an 8% SDS/PAGE gel and electrotransferred onto an Immobilon PVDF membrane (Millipore). Membranes were probed for RNase E as described in Materials and Methods. Size standards are on the left. Asterisks denote the full-length RNase E proteins. (Lane 1) SK9714; (lane 2) SK9937; (lane 3) SK9957; (lane 4) SK9987.

Comparison of the growth properties, mRNA decay rates, and tRNA processing rates of strains carrying the rne⁺, rne-1, rneΔ610, and rneΔ645 alleles in either 6–8 copies/cell or single copy. Growth curves were carried out as described in Materials and Methods. (A) Growth curve with 6–8 copies/cell of each rne allele. (B) Growth curve with each rne allele in single copy. (C) Relative mRNA half-lives as a function of RNase E activity. (D) Relative tRNA precursor half-lives as a function of RNase E activity. The half-life of each mRNA (Table 1) and the half-life of each tRNA precursor obtained with the specific 3′-oligonucleotide probe (Table 3) in the rne-1 strain was set at 1.

Northern analysis of 9S rRNA processing. Five micrograms of total steady-state RNA isolated from either 37°C or 44°C as described in Materials and Methods was separated on a 6% PAGE/7 M urea gel, electrotransferred onto a nylon membrane (MSI MagnaCharge; Osmonics), and probed with a ³²P-5′-end-labeled oligonucleotide complementary to the 5S rRNA (PB5S rRNA). The arrows indicate the 5S rRNA; the larger species are processing intermediates. RQ is the amount of mature 5S rRNA in each strain compared with the wild-type control.

Processing of the glyW cysT leuZ polycistronic transcript. Cultures were grown in LB medium at 44°C as described in Materials and Methods. Ten micrograms of total RNA isolated from SK9714 (rne⁺), SK9937 (rne-1), SK9957 (rneΔ610), and SK9987 (rneΔ645) at 0, 2, 5, 10, 20, and 30 min after rifampicin addition was loaded in each lane. The RNA samples were electrophoresed on a 6% PAGE/7 M urea gel and electrotransferred onto a nylon membrane (MSI MagnaCharge; Osmonics). The same membrane was used for the three Northern blots shown in this figure by following the recommendations of the membrane manufacturer for stripping the probe. (A) Schematic of the glyW cysT leuZ transcript. Numbers indicate the size (in nucleotides) of the spacer regions 5′ and 3′ of the tRNA^Cys gene. Hatched rectangles indicate the oligonucleotide probes. All oligonucleotide probes were 5′-end-labeled with [γ-³²P]ATP using T4 polynucleotide kinase. The diagram is not drawn to scale. (B) Processing of the 3′ end of tRNA^Cys. Northern analysis was done using an oligonucleotide probe (cysT–leuZ) complementary to the 12-nt spacer region between tRNA^Cys and tRNA^Leu. (C) Processing at the 5′ end of tRNA^Cys. The Northern blot was stripped and reprobed with oligonucleotide glyW–cysT complementary to a section of the 54-nt 5′ spacer region of tRNA^Cys. (D) Detection of the mature tRNA^Cys using the cysT probe. Sizes of selected bands are indicated on the right.

Processing of the pheU monocistronic transcript. The same conditions were used as described in Figure 5. (A) Schematic of the pheU monocistronic transcript. The hatched rectangle is the oligonucleotide probe (pheU2) complementary to the mature tRNA^Phe. The diagram is not drawn to scale. (B) Northern analysis using the pheU2 oligonucleotide probe to detect both the mature tRNA^Phe as well as the unprocessed tRNA^Phe precursor. The sizes of both species are indicated on the right.

Processing of the tyrT tyrV tpr polycistronic transcript. The same procedures were followed as described in Figure 5. Hatched rectangles indicate the oligonucleotide probes. (A) Diagrammatic representation of the operon (not to scale). (B) Processing of the 5′-end tyrT. Northern analysis was done using primer tyrT5′A, which was specific for the entire 44-nt 5′ unprocessed terminus of tyrT. (C) Detection of the mature tRNA^Tyr using primer tyrTV3′. The sizes of the various tRNA transcripts are indicated on the right.

Analysis of tRNA processing in RNase E and RNase P mutants. (A) Steady-state RNA was isolated from each strain following a 150-min shift to 44°C as described in Materials and Methods. (B) Total RNA was isolated from cultures shifted to 44°C for the specified times. Five micrograms of total RNA was loaded into each lane of a 6% polyacrylamide/7 M urea gel. Each blot was probed with an oligonucleotide complementary to the mature tRNA. Diagrams of the tRNA species are shown on the left. The sizes on the right were determined using RNA molecular size standards (Invitrogen).

Analysis of steady-state levels of tRNAs in various RNase E mutants. Steady-state RNA was isolated from SK9714 (rne⁺) and SK9937 (rne-1) after cultures had been shifted to 44°C for 120 min as indicated in Materials and Methods. For SK9957 (rneΔ610) and SK9987 (rneΔ645), the cultures were grown for 240 min prior to isolating RNA. The Northern blots were run as described in Figure 5. The membranes were probed with oligonucleotides complementary to either tRNA^His (hisR), tRNA^Pro (proM), tRNA^Asn (asn), or 16S rRNA (rrnB2448). PF denotes the processed fraction that is defined as the amount of a given mature tRNA relative to the total amount of that tRNA (processed and unprocessed). RQ indicates the amount of mature tRNA in each lane compared with that determined in the rne⁺ strain. As a normalization control a blot was probed for 16S rRNA.

Primer extension analysis of tyrT. Two micrograms of total steady-state RNA from SK9714 (rne⁺), SK9937 (rne-1), SK9957 (rneΔ610), or SK9987 (rneΔ645) was subjected to primer extension analysis using a ³²P-end-labeled primer (tyrTV) complementary to a segment of the spacer region between tyrT and tyrV. RNA from SK9714 and SK9937 was isolated after a 120-min shift to 44°C, whereas RNA from SK9957 and SK9987 was isolated after a 240-min shift as described in Materials and Methods. The primer extension product, mapping 1 nt after the CCA terminus (Table 4), is indicated by the arrow. A sequencing ladder obtained by using the same primer is shown at the left.