Article Figures & Data
Figures
- FIG. 1.
Schematic representation of 23S rRNA fragmentation in R. leguminosarum ATCC 10004T.
- FIG. 2.
Mapping of 5′ ends which occur due to processing in the region of helices 9 and 10 of 23S rRNA of R. leguminosarum ATCC 10004T. (A) Model of the potential secondary structure of 23S rRNA positions 109 to 205 (E. coli numbering) in R. leguminosarum ATCC 10004T. H8, H9, and H10, helices 8, 9, and 10, respectively. The positions of primers 23S-IVSI-antisense and Rleg-IVS-2 are marked with lines on the side of the complementary rRNA sequences. The positions of the helix 9 RNase III cleavage sites in vitro and in vivo and of the mature 5′ end of the 2.6-kb rRNA fragment in vivo, as determined by primer extension analysis, are indicated by arrows. Rc, cleavage site of Rc-RNase III in vitro; Ec, cleavage site of Ec-RNase III in vitro; Rl, cleavage site of Rl-RNase III from R. leguminosarum; M, mature 5′ end of the 2.6-kb rRNA fragment in vivo. (B) Determination of RNase III cleavage sites on the left side of helix 9 using primer Rleg-IVS-2. The following RNAs were used as templates in primer extension reactions: 1, unprocessed in vitro transcript; 2, R. leguminosarum total RNA; 3, in vitro transcript cleaved with Rc-RNase III; 4, in vitro transcript cleaved with Ec-RNase III. Lanes G, A, T, and C each refer to the corresponding nucleotide of the DNA template (cloned 23S rDNA region) as determined by sequencing. A part of the rRNA sequence is indicated on the side of the panel. The signals corresponding to the detected 5′ ends are marked by arrows (descriptions as in A). Tr., 5′ end of the unprocessed transcript. (C) Determination of RNase III cleavage sites on the right side of helix 9 using primer 23S-IVSI-antisense. The following RNAs were used as templates in primer extension reactions: 1, in vitro transcript cleaved with Rc-RNase III; 2, in vitro transcript cleaved with Ec-RNase III; 3, in vitro transcript incubated with R. leguminosarum cell extract; 4, R. leguminosarum total RNA; 5, unprocessed in vitro transcript. Lanes G, A, T, and C each refer to the corresponding nucleotide of the DNA template (cloned 23S rDNA region) as determined by sequencing. A part of the rRNA sequence is indicated on the side of the panel. The signals corresponding to the detected 5′ ends are marked by arrows (descriptions as in A). Tr., 5′ end of the unprocessed transcript.
- FIG. 3.
Characterization of the domain III processing site of 23S rRNA in R. leguminosarum ATCC 10004T. (A) Determination of the 5′ end of the distal 1.3-kb rRNA fragment via primer extension analysis. The following rRNAs were used as templates: 1, unprocessed in vitro transcript; 2 and 3, R. leguminosarum total RNA; 4, R. giardinii total RNA. Lanes G, A, T, and C each refer to the corresponding nucleotide of the DNA template (cloned 23S rDNA region) as determined by sequencing. A part of the rRNA sequence is indicated on the side of the panel. The signals corresponding to the detected 5′ end, to the unprocessed transcript (Tr.), and to the primer are marked by arrows. (B) Determination of the 3′ end of the proximal 1.3-kb rRNA fragment via RNase protection analysis. Lanes G, A, T, and C each refer to the corresponding nucleotide of therDNA sense strand as determined by sequencing. R.g., rDNA sequence of R. giardinii H152; R.l., rDNA sequence of R. leguminosarum ATCC 10004T; 1, total RNA of R. giardinii was used for hybridization with the R. giardinii radioactively labeled antisense RNA followed by RNase digestion; 2, R. leguminosarum antisense RNA (RNase protection probe) without RNase; 3, R. leguminosarum antisense RNA incubated with RNase in the absence of total RNA; 4, R. leguminosarum antisense RNA hybridized with total RNA of R leguminosarum and subsequently treated with RNase. A part of the rRNA sequence is indicated on the side of the panel, and the position of the mature 3′ end of the proximal 1.3-kb rRNA fragment is shown by an arrow. Additional signals arising due to the presence of 2.6-kb rRNA in the sample are marked by asterisks. Tr., full-length transcript. (C) Model of the secondary structure of the transcript comprising the analyzed domain III region of 23S rRNA in R. leguminosarum ATCC 10004T (29; 23S rRNA positions 1334 to 1623, E. coli numbering). The positions of the mature ends arising after domain III processing, as determined by primer extension and RNase protection analysis, are indicated by arrows marked 5′ and 3′. Arrows marked with asterisks correspond to the additional signals shown in B. The positions of the oligonucleotides DIIIse2-SQ (used for the sequencing reaction loaded beside the RNase protection analysis samples), 23S-DIII-int1 and 23S-DIII-int2 (used for hybridization), and 23S-DIII-as (used for primer extension) are marked with lines on the side of the complementary rRNA sequences. (D) Northern hybridization of total RNA separated on 1.2% agarose-formaldehyde gels with oligonucleotides 23S-DIII-int1 and 23S-DIII-int2. The size of the detected 23S rRNA fragments is shown on the side of the panel. (E) Schematic diagram showing the relationship between the 2.6-kb and 1.3-kb rRNA fragments, the antisense RNA (RNase protection probe), the oligonucleotide probes 23S-DIII-int1 and 23S-DIII-int2 used for hybridization, and the positions of the signals obtained by RNase protection analysis (marked with arrows, compare with panels B and C). The antisense RNA contains at the 5′ end approximately 50 nucleotides originating from the cloning vector. The sequence complementary to rRNA starts at the position marked by two asterisks.
- FIG. 4.
Ratio of 2.6-kb to 1.3-kb rRNAs changes during growth of R. leguminosarum ATCC 10004T. (A) A representative Northern hybridization of total RNA with the radioactively labeled oligonucleotide 23S-DIII-int1. The time point of harvesting the cells after inoculation of a semiaerobic culture and the amount of total RNA loaded per lane are indicated on the top of the panel. The size of the detected 23S rRNA fragments is shown on the side of the panel.(B) Growth curves of five R. leguminosarum cultures. One of the curves corresponds to the semiaerobic culture analyzed in A. The other four curves show the growth of semiaerobic and aerobic R. leguminosarum cultures inoculated with a fresh inoculum or with the same inoculum kept at 4°C for 72 h (old inoculum) and analyzed in Fig. 3C. (C) Changes in the relative amount of 2.6-kb rRNA during the growth of R. leguminosarum cultures inoculated with fresh or old inoculum. Northern blots were performed with total RNA using the oligonucleotides 23S-IVS-antisense and 23S-DIII-int1. At the following time points after inoculation, samples for isolation of total RNA were harvested: 30 min, 60 min, 90 min, 4 h, 6 h, 8 h, and 24 h. The curves represent the quantitative analysis of the signals obtained. The sum of the intensity of the 1.3-kb and 2.6-kb signals per each lane was defined as 100% of 23S rRNA sequences. The values did not deviate by more than 8%.
