Pyrrolysine Analogs as Substrates for Bacterial Pyrrolysyl-tRNA Synthetasein Vitroandin Vivo

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry

“…The sequence comparison of bacterial PylRS enzymes revealed that L is conserved at this position while archaeal PylRSs share F at the corresponding position (data not shown). An in‐depth analysis of the importance of the F→L mutation for the discrimination of AzF vs Phe by engineered PylRS is currently limited by the fact that only the archaeal Mm PylRS and Mb PylRS (which share 75% sequence identity) as well as the bacterial Dh PylRS are established for SCS.…”

“…The Dh PylRS was found to aminoacylate Dh tRNA CUA Pyl with an entire set of Pyl analogs in vitro . In E. coli , the Dh PylRS/ Dh tRNA CUA Pyl pair accepted different lysine derivatives as the substrate in the order N ϵ ‐[(allyloxy)carbonyl]‐ l ‐lysine (Aloc) > N ϵ ‐(prop‐2‐ynyloxycarbonyl)‐ l ‐lysine (Proc) > N ϵ ‐cyclopentyloxycarbonyl‐ l ‐lysine (Cyc) .…”

“…The Dh PylRS was found to aminoacylate Dh tRNA CUA Pyl with an entire set of Pyl analogs in vitro . In E. coli , the Dh PylRS/ Dh tRNA CUA Pyl pair accepted different lysine derivatives as the substrate in the order N ϵ ‐[(allyloxy)carbonyl]‐ l ‐lysine (Aloc) > N ϵ ‐(prop‐2‐ynyloxycarbonyl)‐ l ‐lysine (Proc) > N ϵ ‐cyclopentyloxycarbonyl‐ l ‐lysine (Cyc) . Thus, the substrate preference is altered compared to the archaeal PylRSs accepting substrates such as N ϵ ‐( tert ‐butoxycarbonyl)‐ l ‐lysine (BocK), Aloc, Proc, and several other Pyl and lysine analogs .…”

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

“…A very sensitive genetic assay revealed weak UAG translation with pylSc, which was not enhanced by co-expression with pylSn (13). A more recent paper indicated that PylSc by itself is capable of supporting fairly high levels of amber codon translation in E. coli (40) Pyl has yet to be formally proven, and it is possible the specificity of tRNA Pyl binding by PylSn and its homologs may increase the fidelity of pyrrolysine incorporation into protein. Even a small error rate in which pyrrolysylation of a noncognate tRNA occurs could result in a deleterious physiological effect.…”

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

“…The mechanism by which A. arabaticum senses TMA and initiates the expression of the Pyl-decoding system remains unknown. Interestingly, despite encoding a functional PylRS 105,106 and tRNA Pyl pair 107 , Desulfitobacterium hafniense did not show detectable production of Pyl-tRNA Pyl or expression of MttB. Desulfitobacterium dehalogenans expressed transcripts for tRNA Pyl and PylRS, but no detectable transcription of the Pyl biosynthesis genes ( pylB , pylC and pylD ) was observed.…”