UC San Diego

N6 -threonylcarbamoyladenosine (t6A) is a universally conserved tRNA modification found at position 37 of ANN-decoding tRNAs (N is A, U, G or C) and is essential for accurate and efficient translation. t6A37 biosynthesis occurs in two steps: synthesis of the pathway intermediate threonylcarbamoyladenylate (TC-AMP) by TsaC/TsaC2 enzymes, followed by transfer of the threonylcarbamoyl moiety onto A37 of substrate tRNAs, catalyzed in bacteria by the threonylcarbamoyl transfer (TCT) complex. Although the enzymes responsible for t6A37 biosynthesis have been identified and characterized biochemically, key mechanistic questions have remained unresolved, including how TsaC2 catalyzes TC-AMP formation and what drives substrate tRNA recognition. In this dissertation, I used X-ray crystallography, SEC-SAXS, cryo-EM, and biochemical assays to investigate t6A biosynthesis in Thermotoga maritima. In Chapter 2, I performed time- and pH-dependent soaking of TmTsaC2 crystals to capture substrate-, intermediate-, and product-bound states of the enzyme. These structures provide direct evidence for N-carboxy-L-threonine as a reaction intermediate. In addition, SEC-SAXS analysis revealed that TmTsaC2 adopts a closed conformation in the presence of substrates in solution but exhibits high conformational flexibility in their absence. In Chapter 3, I used cryo-EM to determine structures of the Thermotoga maritima t 6A synthase bound to unmodified and t6A-modified tRNA. These structures reveal that productive tRNA binding requires dramatic remodeling of the anticodon loop into a zig-zag conformation. tRNA is recognized through an indirect readout mechanism in which the key t6A determinants, U36 and A38, form a base triple with U32 instead of making base-specific interactions with the protein. The tRNA D-stem is also recognized predominantly by shape rather than sequence-specific contacts. Together, these data fill important knowledge gaps in the t6A field by defining key steps in TC-AMP synthesis and revealing how the bacterial t 6A synthase selects substrate tRNAs for modification. This work provides a structural and mechanistic foundation for future studies of this essential tRNA modification pathway.