Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.

The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another. The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition. Here we report that aminoacylation of Escherchia coli tRNACYS depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48. While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E. coli tRNACYS has guanosine -15 and -48. Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation. Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA. This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and 0-6. Our results provide evidence for a role of RNA tertiary structure in synthetase recognition.All transfer RNAs (tRNAs) fold into a cloverleaf structure that consists of four double-helical stems and four singlestranded regions known as the dihydrouridine (D), anticodon, extra (or variable), and T'IC loops, where T is pseudouridine. Within this cloverleaf, a set of conserved and semiconserved nucleotides establish a network of tertiary interactions that fold the cloverleaf into an "L"-shaped tertiary structure. In this structure (Fig. 1), the amino acid acceptor stem stacks directly on the TTC stem to form one arm of the L structure, while the D stem stacks on the anticodon stem to form the other arm of the L. The two arms are thenjoined by tertiary interactions between the D and the TPC loops and between the D and variable loops so that the 3' CCA sequence and the anticodon are placed at the opposite ends of the L structure (1, 2). The two ends are separated by about 75 A. Various locations scattered along the inside ofthe L tertiary structure have recently been implicated as sites for tRNA recognition by aminoacyl-tRNA synthetases (3-6), which are the group of enzymes that catalyze the specific attachment of an amino acid to the CCA end.The most frequently observed interactions between a tRNA and its cognate synthetase are with the anticodon, the acceptor helix, and the discriminator base (nucleotide 73) adjacent to the CCA end (3-6). In Escherichia coli, the alanyl-and histidinyl-tRNA synthetases primarily recognize determinants in the acceptor helix, while the methionyl-, isoleucyl-, and valyl-tRNA synthetases depend largely on the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertis...

Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis.

Bacterial multi‐drug resistance is a burgeoning crisis whose origin remains mechanistically opaque. Through altered permeability or efflux activity, the membrane prevents many antibiotics from reaching high enough intracellular concentrations to exert a therapeutic effect. We show here that protein synthesis of membrane‐associated genes involves translation of proline CC[C/U] codons, which require N1‐methylation of guanosine 37 (m1G37) on the 3′‐side of the tRNA anticodon. TrmD is the bacterial methyl transferase that catalyzes m1G37‐methylation. Removal of m1G37 by trmD inactivation reduces biosynthesis of membrane proteins, impairs membrane structure and mechanics, and sensitizes Gram‐negative bacteria to multiple classes of antibiotics and suppresses development of resistance and persistence. Codon engineering in the major efflux gene tolC removes the dependence on trmD for biosynthesis of membrane proteins. TrmD activity requires the conserved G37 in tRNAPro; replacement of G37 with C37 increases membrane permeability and accelerates cell death. These findings demonstrate the potential to diminish multi‐drug resistance by inaction of TrmD and its m1G37‐tRNA product. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.