Figure 5.

5′-RACE analysis of B. subtilis trnSL-Ala1 3′-precursors. (A) Secondary structure of tRNA^Ala1 primary transcripts with RNase P and Z cleavage sites indicated; the arrow aligning the transcription terminator and tRNA acceptor stem indicates the position of the primer (‘180’) used for the 5′-RACE experiment; the boxed nucleotides (U74, A75) are replaced with the two C residues after RNase Z cleavage and CCA addition. (B) Analysis of 5′-RACE RT-PCR products on a native 8% PAA gel; M, 10-bp ladder as size marker; in lanes 1–3, primer ‘180’ (see above) and in lanes 4–6  a primer (see the Materials and Methods section) specific for T. thermophilus tRNA^Gly (Figure 1D) was used. The arrow on the left indicates the main amplification product obtained with primer ‘180’; its size was expected for a tRNA^Ala1 processing intermediate with a mature 5′-end but carrying the entire 3′-extension as in the primary transcript; this type of product was only detected in total RNA isolated from SSB320 cells grown in the absence of IPTG (lanes 1 and 2), but not in the presence of IPTG (lane 3); before reverse transcription, total RNA preparations were treated (+TAP) or not treated (− TAP) with tobacco acid pyrophosphatase (TAP). In lanes 5 and 6, total RNA from SSB320 cells grown in the absence of IPTG was supplemented with in vitro transcribed ptRNA^Gly (Figure 1D; 0.05 A₂₆₀ units ptRNA^Gly added to 1.0 A₂₆₀ unit of total cellular RNA) starting with 5′-pppG. A product corresponding to the size of ptRNA^Gly was only detected after TAP treatment (lane 6), but not without TAP treatment (lane 5) or without addition of in vitro transcribed tRNA^Gly (lane 4), thus confirming that TAP was active.