Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA‐modifying enzyme, archaeal tRNA‐guanine transglycosylase

Abstract

In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ 0 ). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ 0 . We prepared a series of hypomodified tRNAs Ser (GGA) from Escherichia coli strains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAs Ser (GGA) as substrates showed that the K m decreased due to the lack of modified nucleosides. The tRNAs Ser (GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNASer (GGA) enhanced tRNA stability. Furthermore, the ArcTGT K m strongly correlated with the melting temperature (T m ), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ 0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.

tRNA Modifications: Impact on Structure and Thermal Adaptation

“…Furthermore, a strong metal binding site in cytosolic tRNAs was identified at position G15 in the D-loop [96], where a hydrated Mg 2+ stabilizes a reverse Watson-Crick base pair G15-C48 (the Levitt base pair [97]) between D-loop and variable loop [98]. In archaea, a comparable stabilization of the Levitt base pair is achieved by a modification of G15, leading to archaeosine (G+; 7-formamidino-7-deazaguanosine, see Figure 2) [99,98]. While this archaea-specific modification interferes with Mg 2+ binding, the positively charged formamidine group at the C7 atom fulfils the same function as the coordinated Mg 2+ and stabilizes the tRNA structure in a similar way [98].…”

“…While the stability and (non-)isostericity of individual base pairs (e.g., GC versus GU) can increase the local flexibility in a tRNA domain [137], there are also modifications that contribute to the conformational elasticity of these molecules. Dihydrouridine (D, see Figure 3C for a three dimensional structure) is the name-giving modification that is frequently found in the D-loop, predominantly at positions 16, 17, 20, 20a and 20b, and additionally at position 47 in the variable loop [18,27,99,138,139,140,141,142,143]. It is formed by a reduction of the double bond between positions C5 and C6 in the pyrimidine ring of uridine [144].…”

“…Nevertheless, D can also induce stability in a neighboring helical region, as shown in the D-arm of Schizosaccharomyces pombe tRNA i Met , where D enhances the flexibility of the D-loop, while it simultaneously forces the D-stem to adopt a rather stable conformation [41]. Yet, it came as a surprise that the lack of D20a in the E. coli tRNA Ser lowered the melting temperature, indicating that the presence of this modification contributes to tRNA stability [99]. It is discussed that the D-mediated locally increased flexibility may facilitate the formation of the stabilizing neighboring tertiary base interactions in the elbow region of the tRNA.…”

“…It is discussed that the D-mediated locally increased flexibility may facilitate the formation of the stabilizing neighboring tertiary base interactions in the elbow region of the tRNA. In the same series of experiments, it was shown that these tertiary interactions are then further stabilized by the modification of the involved bases like the above mentioned s 4 U8 that pairs with A14 in the D-loop (Figure 1) [99]. …”

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

“…Their biological roles often extend beyond providing immunity against restriction enzymes 1,4,6,46 to those concerning regulation of gene expression (base J in trypanosomes 47,48 , hydroxymethylcytosine 49 ), protein synthesis (e.g. tRNA anti-codon loop modifications [50][51][52][53], and structural and thermal stability of nucleic acids [54][55][56] . As in the case of Mom, biosynthetic pathways for several modifications have long remained enigmatic until recently, chemistries underlying some began to be unraveled 26,33,45,57,58 .…”