Article Figures & Data
Figures
- FIG. 1.
Neighbor-joining phylogenetic tree for the bacterial NTSF enzymes. The tree was constructed using PHYLIP. Bootstrap scores are shown at the nodes. The branch lengths do not represent evolutionary distances. The species represented are E. coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, Pseudomonas aeruginosa, D. radiodurans, A. aeolicus, Nostoc spp., Synechocystis spp., G. sulfurreducens, G. metallireducens, P. carbinolicus, and D. psychrophila.
- FIG. 2.
(A) RT-PCR analysis showing the expression of the genes encoding G. sulfurreducens NTSF enzymes. Lane 1, size standards (the arrow indicates the position of the 1-kb standard); lanes 2, 4, and 6, RT-PCR products obtained with primers designed to identify NTSFI, NTSFII, and NTSFIII, respectively; lanes 3, 5, and 7, PCR products obtained with the same primers but without RT. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of purified NTSFI (1 μg) (lane 2), NTSFII (2.5 μg) (lane 3), and NTSFIII (2.5 μg) (lane 4). Lane 1 contained size standards having molecular weights ranging from 20,000 to 106,000. The arrow indicates the position of the 50,000-M r standard.
- FIG. 3.
Autoradiogram of denaturing polyacrylamide gels of the reaction products from assays of E. coli PAP I (lane 3), G. sulfurreducens (G. sulf.) NTSFI (lane 4), S. coelicolor (S. coel.) SCO3896 with CTP and tRNAAsp-C as substrates (lane 5), SCO3896 with ATP and tRNAAsp-CC as substrates (lane 8), NTSFII with CTP and tRNAAsp-C as substrates (lane 6), NTSFII with ATP and tRNAAsp-CC as substrates (lane 7), NTSFIII with ATP and tRNAAsp-CC as substrates (lane 9), and NTSFIII with CTP and tRNAAsp-C as substrates (lane 10). Lanes 1 to 4 are lanes from a 5% polyacrylamide gel, and lanes 5 to 11 are lanes from a 10% polyacrylamide gel. Lanes 1 and 11 contained end-labeled size markers (STDS). For comparison, lane 2 shows the migration position of the end-labeled rpsO-pnp transcript, but it should be noted that unlabeled transcript was used in the polyadenylation assays. The rpsO-pnp transcript migrated more rapidly than expected, presumably due to the presence of some secondary structure in the transcript even in the presence of urea (20).
- FIG. 4.
Measurement of the lengths of oligonucleotide and poly(A) stretches found in the 3′ tails of RNAs from E. coli DH5α (lane 2), B. subtilis (lane 3), B. halodurans C-125 (lane 4). and G. sulfurreducens (lane 5). Lane 1 contained an oligo(dT18) size standard. Labeled 3′ tails were fractionated on a 12% denaturing polyacrylamide gel.
- FIG. 5.
Positions and compositions of G. sulfurreducens RNA 3′ tails. The position of each tail was determined from the mature 5′ end of rRNA gene or the translation start site of the NTSF enzyme. The number of the last nucleotide of the mature rRNA or translation stop codon is indicated at the top right. The figure is not drawn to scale.
