Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: The 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA

Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation

“…Total RNA for northern blots was prepared using glass beads as described (Ausubel et al+, 2001) Figure 6+ To detect tRNA i Met , the oligonucleotide 59-GGACATCAGGGTTATGA GCC-39 was used+ Oligonucleotides were labeled using adenosine 59[g 32 P]-triphosphate (5,000 Ci/mmol, Amersham) and polynucleotide kinase (Roche Molecular Biochemicals)+ Northern blots were visualized and quantified by PhosphorImager analysis+ Total tRNA for HPLC analysis was prepared essentially as described (Nordlund et al+, 2000) except for the 5-min vortexing step, which was shortened to 2 min+ The tRNA elution buffer was 0+8 M NaCl, 50 mM Tris-HCl, pH 8+0, 15% ethanol+ For HPLC analysis, 50 mg tRNA were digested to nucleosides using nuclease P1 and bacterial alkaline phosphatase (Gehrke et al+, 1982) and the hydrolysate was analyzed by HPLC (Gehrke & Kuo, 1990) using a Waters TM System liquid chromatograph with a Waters TM 996 diode array UV detector (Waters TM Corporation, Milford, Massachusetts, USA)+ Separation of nucleosides was achieved using a Supelcosil LC-18-S reverse-phase column (4+6 by 250 mm) and a Supelguard LC-18-S, 2+1 by 20 mm guard column (Supelco, Bellefonte, Pennsylvania, USA) thermostatted to 26 8C, at a flow rate of 1 mL/min+ For MS analysis, the flow rate was 2 mL/min, and the UV detector was directly interfaced to a VG platform mass spectrometer equipped with an electrospray ionization source (Fisons Instruments, Altrincham, UK)+ In MS analysis, nucleosides were eluted using the gradient of Buck et al+ (1983)+ UV data were recorded continuously, and mass spectra were recorded every 1+0 s during the 60-min chromatography+ The procedures and interpretation of data for qualitative LC-MS analysis of nucleosides in RNA hydrolysates have been previously described in detail (Pomerantz & McCloskey, 1990)+…”

Substitutions in an Active Site Loop of Escherichia coli IscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesis in Vivo

“…All of the thionucleosides were analyzed at 260 nm with the exception of 4-thiouridine, which was also analyzed at 330 nm (data not shown). To account for variation in sample loading, the thiouridines and s 2 C were quantitated by taking the ratio of each peak area to that of pseudouridine in the same chromatogram (41). The levels of ms 2 i 6 A were taken as a relative percentage of the sum of ms 2 i 6 A and i 6 A, which should be independent of the amount of tRNA loaded onto the column.…”

Enzymatic Modification of tRNAs

“…Iron was determined by the method of Fish (22), and inorganic sulfide was quantified spectrophotometrically as described by Beinert (23). Modified nucleosides were analyzed from tRNAs isolated as described previously (18,24). tRNAs were digested to nucleosides with nuclease P1 and alkaline phosphatase.…”