Abstract
The anticodon stem-loop (ASL) of transfer RNAs (tRNAs) drives decoding by interacting directly with the mRNA through codon/anticodon pairing. Chemically complex nucleoside modifications found in the ASL at positions 34 or 37 are known to be required for accurate decoding. Although over 100 distinct modifications have been structurally characterized in tRNAs, only a few are universally conserved, among them threonylcarbamoyl adenosine (t(6)A), found at position 37 in the anticodon loop of a subset of tRNA. Structural studies predict an important role for t(6)A in translational fidelity, and in vivo work supports this prediction. Although pioneering work in the 1970s identified the fundamental substrates for t(6)A biosynthesis, the enzymes responsible for its biosynthesis have remained an enigma. We report here the discovery that in bacteria four proteins (YgjD, YrdC, YjeE, and YeaZ) are both necessary and sufficient for t(6)A biosynthesis in vitro. Notably, YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development.
Highlights
The modified nucleoside t6A is important for transfer RNAs (tRNAs) function
To test the hypothesis that t6A biosynthesis in E. coli requires YgjD—Cultures of transformed cell lines containing pBY215.1 (YrdC), YgjD, YeaZ, and YjeE, the yjeE and yeaZ genes from E. coli were expressed as N-terminal His6-fusion proteins similar to our construct for yrdC expression [10]
Several RNAs were employed as potential substrates, including unfractionated yeast tRNA from a ⌬sua5 mutant [18], unmodified E. coli tRNAThr and tRNALys transcripts produced through in vitro transcription, and an unmodified stem-loop RNA corresponding to the anticodon stem-loop (ASL) of E. coli tRNALys, which has recently been shown to bind YrdC [26]
Summary
The modified nucleoside t6A is important for tRNA function. Results: The proteins YrdC/YgjD/YeaZ/YjeE are necessary and sufficient for the biosynthesis of t6A in bacteria. YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development. Comparative genomic and genetic studies linked the YrdC/Sua5 [18] and YgjD/Kae1/ Qri7 [10, 19] families to t6A biosynthesis Both families are universally conserved and were ranked among the top 10 proteins of unknown function in need of functional characterization [20]. These observations together with the strong physical clustering of ygjD, yeaZ, and yjeE genes [10, 21] led us to investigate whether YeaZ and YjeE might be involved in t6A biosynthesis in bacteria
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
