Maturation Pathways for E Maturation Pathways for E. coli tRNA Precursors: A Random Multienzyme Process In Vivo  Zhongwei Li, Murray P Deutscher  Cell  Volume 86, Issue 3, Pages 503-512 (August 1996) DOI: 10.1016/S0092-8674(00)80123-3

Figure 1 Northern Analysis of tRNATyr1 and tRNAArg2 at Low Resolution Total RNA samples were isolated from 14 ml of culture as described in Experimental Procedures and dissolved in gel loading buffer in a volume of 240 μl per A550 of culture. Ten μl of RNA sample was subjected to electrophoresis and Northern analysis as in Experimental Procedures except that the xylene cyanol dye was run for only 7 cm. Hybridization was at 37°C for each tRNA. (A) tRNATyr1; (B) tRNAArg2. Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)

Figure 2 Northern Analysis of the tRNATyr Family Total RNA was prepared as described for Figure 1. For analysis of tRNATyrsu3+, cells contained the single copy plasmid, pOUCCA. Four μl of sample, containing 6–9 μg of RNA, was subjected to electrophoresis as in Figure 1 except that the xylene cyanol was allowed to migrate 29 cm to give single nucleotide resolution. The bottom 8 cm of the gel, containing the tRNA-size molecules, was transferred to a membrane and analyzed by Northern blotting as described in Experimental Procedures. Hybridization was at the temperatures indicated below, and membranes were washed at increasing temperatures to eliminate nonspecific signals. The oligonucleotide probes used are described in Experimental Procedures. Commercial tRNATyr (0.2 μg) was used as a size standard, and its position (M) is indicated. (A) tRNATyr1, hybridization at 37°C; (B) tRNATyr2, hybridization at 25°C; (C) tRNATyrsu3+, hybridization at 25°C. Strain CA244 lacking the pOUCCA plasmid was used as the negative control (lane 1). Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)

Figure 3 Northern Analysis of tRNATyr1, Expressed from a Multicopy Plasmid Total RNA was prepared as described for Figure 1 from strains carrying the plasmid pBRTyrT. Analysis was carried out as described for Figure 2 using hybridization at 37°C. The size of mature tRNATyr1 (M) is indicated. Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)

Figure 4 Southern Analysis of the Genes for tRNATyr1 and tRNATyrsu3+ Total DNA was isolated from strains carrying either pBRTyrT (A) or pOUCCA (B). Approximately 10 μg of DNA digested wih PvuII was loaded for each sample and analyzed as described in Experimental Procedures. Hybridization with the tRNATyr1-specific oligonucleotide, which detects both the chromosomal and plasmid-borne copies of the tyrT and su3+ genes, was carried out at 37°C. DNA from strain CA244 (WT) and CA244ΔTyrT (ΔTyrT) served as positive and negative controls for the chromosomal band. Arrows on the left indicate the positions of the chromosomal (chr) and plasmid (pl) genes. Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)

Figure 5 Northern Analysis of the tRNAVal Family Total RNA was prepared and analyzed as in Figure 1 and Figure 2. Gels were run until the xylene cyanol marker had migrated 26 cm. Hybridization was carried out at the temperatures indicated using the oligonucleotide probes described in Experimental Procedures. (A) tRNAVal1, 37°C; (B) tRNAVal2B, 44°C; (C) tRNAVal2B, 37°C. Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)

Figure 6 Synthesis of tRNATyr1 and rtT mRNA in Cells Lacking Both RNase II and PNPase Cells were grown in Luria Broth supplemented with glucose (0.2%) and ampicillin (50 μg/ml) at 30°C. At time 0 the temperature of the cultures was shifted to 45°C and samples were withdrawn at various times to measure growth (A), RNase II activity (B) and the levels of tRNATyr1 (C) and rtT mRNA (D). RNA samples at each time point were prepared and analyzed as in Figure 1. Hybridization was at 37°C for tRNATyr1 and at 42°C for rtT mRNA with the probes described in Experimental Procedures. Cell 1996 86, 503-512DOI: (10.1016/S0092-8674(00)80123-3)