The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli  Kei Kitahara, Tsutomu Suzuki  Molecular Cell  Volume 34, Issue 6, Pages 760-766 (June 2009) DOI: 10.1016/j.molcel.2009.05.014 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Construction of CPs of 16S and 23S rRNAs Schematic diagrams of the domain structures of five CPs (cp33, CP45, CP63, CP63+PS, and CP78) shown in the E. coli rrnB operon. Four domains of the 16S rRNA—the 5′ domain (5′), the central domain (C), the 3′ major domain (3′M), and the 3′ minor domain (3′m)—and six domains of the 23S (domains I to VI) rRNA are colored. Terminal RNA helices of CPs and cleavage sites for RNaseIII and maturases (M16 and M23) are indicated with small vertical arrows. The 5′- and 3′-processing stems for 16S (5′-ps and 3′-ps) and 23S (5′-PS and 3′-PS) are shown as circles. Locus and direction of DNA primers (Table S1) for RT-PCR analyses are indicated with horizontal arrows. These CPs were constructed in the plasmid pRB103 (pSC101 ori), which encodes rrnB and the zeocin (zeo) resistant marker. Molecular Cell 2009 34, 760-766DOI: (10.1016/j.molcel.2009.05.014) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Analyses of rRNAs in CPs of 16S and 23S rRNAs (A) Analysis of rRNAs transcribed from CPs, using denaturing agarose gel electrophoresis. Total RNAs extracted from actively growing WT KT103 cells and of KT103 cells transformed with cp33, cp33/ΔdeaD, CP45, CP63, CP78, and CP63+PS were electrophoresed on a 1.2% formaldehyde agarose gel. The gel was stained with ethidium bromide. The identity of each band is indicated next to an arrowhead. (B) Confirmation that the rRNAs were transcribed from circular permutants, using RT-PCR analysis. Total RNAs from WT KT103 cells and from KT103 cells transformed with CPs were subjected to RT-PCR using 806F and 1176R, 1405F and 121R, 1131F and 2175R, and 2754F and 126R primer sets (Figure 1A and Table S1) to amplify cDNAs of 371 bp, 374 bp, 1,045 bp, and 279 bp (318 bp for CP63+PS), respectively. The sizes of the DNA maker fragments are indicated. The cDNAs were electrophoresed and stained with ethidium bromide. Molecular Cell 2009 34, 760-766DOI: (10.1016/j.molcel.2009.05.014) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Growth Properties and Sucrose Density Gradient Centrifugation Profiling of the CPs (A) Growth properties of KT103 cells transformed with CPs. Overnight full-growth culture of KT103 and KT103/ΔdeaD cells expressing WT, cp33, CP45, or CP63 rRNA were serially diluted (103–106 fold dilution), were spotted onto zeosin and sucrose-containing LB plates, and were cultivated at 37°C for 22 hr. The doubling time (Table 1) of each mutant in SOB liquid medium is shown on the right side of each photograph. (B) Growth properties of CP45/ΔdeaD cells transformed with pBAD-deaD. An empty vector of pBAD (mock) was employed as a negative control. The doubling time (Table 1) is shown for each photograph. (C) SDG profiling of ribosomal subunits in the cell lysates of CPs in the presence of 6 mM (left panels) or 0.5 mM (mid and right panels) of Mg2+. Peak assignments of the 70S, 50S, and 30S subunits are indicated in the profile. Profiles of WT KT103 and of KT103 transformed with CPs are shown in black lines. Profiles of KT103/ΔdeaD and of KT103/ΔdeaD transformed with CPs are shown in red lines (only in the mid and right panels). Molecular Cell 2009 34, 760-766DOI: (10.1016/j.molcel.2009.05.014) Copyright © 2009 Elsevier Inc. Terms and Conditions