Most cell cycle-regulated genes adopt non-optimal codon usage, namely, their translation involves wobbly matching codons. Here, the authors show that tRNA expression is cyclic and that codon usage, therefore, can give rise to cell-cycle regulation of proteins.

TrmD and Trm5 are respectively the bacterial and eukarya/archaea methyl transferases that catalyze transfer of the methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 in tRNA to synthesize m1G37-tRNA. The m1G37 modification prevents tRNA frameshifts on the ribosome by assuring correct codon-anticodon pairings, and thus is essential for the fidelity of protein synthesis. Although TrmD and Trm5 are derived from unrelated AdoMet families and recognize the cofactor using distinct motifs, the question of whether they select G37 on tRNA by the same, or different, mechanism has not been answered. Here we address this question by kinetic analysis of tRNA truncation mutants that lack domains typically present in the canonical L shaped structure, and by evaluation of the site of modification on tRNA variants with an expanded or contracted anticodon loop. With both experimental approaches, we show that TrmD and Trm5 exhibit separate and distinct mode of tRNA recognition, suggesting that they evolved by independent and nonoverlapping pathways from their unrelated AdoMet families. Our results also shed new light onto the significance of the m1G37 modification in the controversial quadruplet-pairing model of tRNA frameshift suppressors. KeywordstRNA(m1G37) methyl transferase; anticodon stem-loop; frameshift suppressor tRNA; m1G37 tRNAs in all organisms contain extensive site-specific base and backbone modifications that are important for their overall structural stability and functions 1; 2; 3 . Many of these modifications are in the anticodon region to improve tRNA-ribosome interactions and enhance translational fidelity. A notable example is the m1G37 modification that is created by introducing a methyl group to the N1 position of the G37 base 3′ to the anticodon. This modification occurs almost universally in tRNA species that contain G37 and is found in all three domains of life. It is also present in organelles (mitochondria and chloroplasts) and in the bacteria Mycoplasma spp, which have the smallest genomes known to date 4 . The presence of the m1G37 modification has been reported by previous genetic studies to increase ribosomal selectivity of tRNAs at the A site 5 and to reduce frameshift errors during translation 6; 7 . A structural analysis suggests that the m1G37 modification imposes constraints on the anticodon loop dynamics 8 , providing a rationale for the ability of the modified base to enforce codonanticodon pairings. The importance of the m1G37 modification in the overall quality of protein synthesis emphasizes its conservation throughout evolution. ; Email: Ya-Ming.Hou@jefferson.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discover...

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.

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. While both TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3' position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor, TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinities to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed "half-of-the-sites" reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel to those between the two classes of aminoacyl-tRNA synthetases, which use distinct active-site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.