The pyl genes from the methanogenic archaeon M. acetivorans (Ma) and the Gram-positive Bacterium D. hafniense (Dh). The gene order of the pyl genetic code expansion cassette is conserved, with the exception that the D. hafniense pylS gene homolog has been split into two genes encoding homologs to the PylS N-terminal domain (pylSn) and the catalytic core domain (pylSc) that now flank pylBCD (7).

Cells bearing pylB, pylC, and pylD produce pyrrolysine detectable by in vitro assays using pyrrolysyl-tRNA synthetase. (A) The direct aminoacylation of tRNA^Pyl with pyrrolysine present in cell extracts by PylS. Charged and uncharged tRNA species in the isolated cellular pool of tRNA^Pyl were separated in an acid-urea polyacrylamide gel that was subsequently electroblotted. The blot was then probed with ³²P-labeled oligodeoxynucleotide complementary to tRNA^Pyl and analyzed by phosphorimager. Lane 1 was loaded with cellular tRNA as isolated (9) and has both charged (upper arrow) and uncharged (lower arrow) tRNA^Pyl, whereas lane 2 is the cellular tRNA pool following deacylation at pH 9 for 30 min and shows only uncharged tRNA^Pyl. Aminoacylation of tRNA^Pyl by PylS with pyrrolysine was then tested in 25-μl reactions that contained: 3.2 μM PylS, 50 mM KCl, 1 mM MgCl2, 5 mM ATP, 0.5 mM DTT, 8 μg of M. acetivorans deacylated cellular tRNA, and the metabolite pool from the indicated E. coli strains in 10 mM Hepes buffer, pH 7.2. After incubation for 40 min at 37°C, aminoacylation was tested as above in reactions that also contained the following: lane 3, no metabolite pool; or the pool from, lane 4, pK13 bearing pylB, pylC, and pylD; 5, pK14 bearing pylB and pylD; 6, pK15 bearing pylC and pylD; 7, pK16 bearing pylB and pylC; and 8, pACYCDuet-1 bearing no pyl gene. (B) Cellular amino acid pools were tested for activity in the pyrophosphate:ATP exchange assay mediated by PylS in the presence of pyrrolysine. Exchange of ³²P-pyrophosphate into ATP was monitored in 100-μl reactions containing 5.5 μM PylS, 10 mM MgCl₂, 25 mM KCl, 1 mM potassium fluoride, 4 mM DTT, 2 mM ATP, and 2 mM ³²P-PPi (12 dpm/pmol) in 20 mM Hepes-KOH (pH 7.2) and incubated at 37°C. Aliquots were removed at the time points indicated, and the amount of radiolabel bound to acid-washed activated charcoal was quantified to estimate the amount of ³²ATP formed. Shown are illustrated results from averaged duplicate reactions that were supplemented with the extracted amino acid pool from pK13 (●), pK14 (X), pK15 (△), pK16 (◇), or pACYCDuet-1 (○).

UAG translation in mtmB1 in E. coli bearing the five pyl genes. SDS-solubilized extracts of E. coli transformed with pylT, pylS, and mtmB1 on pDLBAD were analyzed by anti-MtmB1 immunoblot. Additionally, the strains were also transformed with the following: lane 1, the vector pACYCDuet-1; lane 2, pK13 bearing pylB, pylC, and pylD; lane 3, pK14 bearing pylB and pylD; lane 4, pK15 bearing pylC and pylD; and lane 5, pK16 bearing pylB and pylC. Lane 6 contains a set of standard proteins with molecular masses indicated at the right in kilodaltons, whereas lane 7 contains purified MtmB1. The upper arrow indicates the location of the mtmB1 UAG translation product, and the lower arrow indicates the location of the mtmB1 UAG termination product.

Mass spectrometry demonstrates that the UAG-encoded residue of recombinant mtmB1 is pyrrolysine in cells bearing the five pyl genes. (Inset) An anti-MtmB1 immunoblot of the 7-M urea-solubilized fraction from the cell pellet of French-press-lysed cells transformed with pDLBAD and either pACYCDuet-1 or pK13, as indicated. The upper arrow points to the UAG-translation mtmB1 product, whereas the lower arrow points to the UAG-termination product. Subsequently, the 7-M urea fraction was electrophoresed in an SDS-polyacrylamide gel, and the 50-kDa MtmB1 band was excised and subjected to in-gel digestion with chymotrypsin and analysis by mass spectrometry. The predicted sequence of the peptide ion at m/z = 783.43²⁺ from chymotryptic cleavage of MtmB1 is shown along with the fragmentation sites forming the a-, b-, and y-series ions identified in the ion's collision-induced dissociation spectrum shown below. The peak of the parent ion (PI) is indicated. A horizontal arrow lies between the a-, b-, or y-series ion pairs whose mass differences allow calculation of the mass of the pyrrolysyl residue. The masses of the remaining identified ion peaks are listed in SI Table 1.