Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA Gayathri Srinivasan, Carey M. James, and Joseph A. Krzycki

The pylT gene was first identified by using the tRNAScanSE program maintained at the Washington University Department of Genetics (http://www.genetics.wustl.edu/eddy/tRNAscan-SE/) and the partially sequenced genome of M. barkeri Fusaro. The program was run in the Cove only mode. The folded tRNACUA was assigned a COVE score of 29.2. Similarly, the tRNACUA in Desulfitobacterium hafniense was first found by identity searches of M. barkeri tRNACUA against D. hafniense genomic sequence (GenBank DOE_49338) followed by tRNAscan-SE folding of the contig containing the putative tRNACUA from the Gram-positive organism.

Chromosomal DNA was isolated from M. barkeri MS as described in Paul et al. (S1), and pylT, pylS, pylB, all but the last 18 codons of pylC amplified by PCR reaction using primers designed with the M. barkeri Fusaro sequence. Automated sequencing was as in (S1). The two sequenced pylT gene clusters of the two strains were 93% identical over their entire length.

In order to produced recombinant PylS, the M. barkeri MS pylS gene was PCR amplified using chromosomal DNA as template and 18-nucleotide-long oligonucleotide primers identical to the 5' and 3' ends of the M. barkeri Fusaro pylS gene. The pylS gene was cloned into pET15b (Novagen, Madison, WI) to create pXRS and then transformed into Escherichia coli BL21(DE3)plysS. The correct insertion of the M. barkeri MS pylS gene in pET15b was confirmed by sequencing of pXRS. One liter of E. coli transformed with pXRS was grown overnight in the presence of IPTG. The cells were resuspended in 300 mM NaCl in 50 mM sodium phosphate, pH 7.4, and broken in a French pressure cell. The centrifuged cell extract supernatant (12 mg protein/ml) was loaded onto a 1 ml HiTrap column (Pharmacia-Amersham, Piscataway, NJ), then washed with 10 ml of buffer containing 300 mM NaCl, 10 mM imidazole, in 50 mM sodium phosphate, pH 7.4. Recombinant PylS immediately eluted from the column with application of 500 mM imidazole in the same buffer, yielding 3 mg of PylS. Control experiments were performed identically, but with an equivalent amount of extract protein from E. coli transformed with pET15b lacking the pylS insert. The molecular weight of the sole polypeptide found in the imidazole eluate of E. coli transformed with pXRS was 48.8 ( 1.1 kD polypeptide, which agreed well with the 48.5-kD molecular mass calculated for PylS bearing the hexahistidine sequence.

Log phase M. barkeri MS was harvested and the tRNA pool isolated by the procedure of Curnow et al. (S2) with the exception that the DEAE step was omitted. This resulted in a preparation enriched with tRNA, but still having some higher molecular weight RNA as judged by ethidium staining of the sample following denaturing agarose electrophoresis.

Supplemental Figure 1. The pylT gene cluster in Desulfitobacterium hafniense. (A) ORFS found in this Gram-positive bacterium on contig 3273 in GenBank DOE_49338 with homology to the pyl genes found in M. barkeri. (B) The secondary structure of the predicted tRNACUA produced from the D. hafniense pylT gene. The sixth base pair in the anticodon stem loop is indicated by two dashes.

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Supplemental Figure 2. FastA files of pyl gene protein products from M. barkeri Fusaro and D. hafniense. These are deduced from preliminary genomic DNA sequence found in GenBank DOE_49338 (D. hafniense) and GenBank NC_002724 (M. barkeri Fusaro).

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References

S1. L. Paul, D. J. Ferguson, J. A. Krzycki, J. Bacteriol. 182, 2520-2529 (2000).
S2. A. W. Curnow, D. L. Tumbula, J. T. Pelaschier, B. Min, D. Söll, Proc. Natl. Acad. Sci. U.S.A. 95, 12838-12843 (1998).