Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl- N ⁶ -(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

Abstract

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TWC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNAMet the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the Vmax value is decreased by onehalf. (ii) tRNAfmet species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNAPet species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated MetAtRNAmet species, however, differ from each other: "Met-tRNAm't rT" is inactive, whereas the U54 counterupart effectively forms the initiation complex. (iii) Two isoacceptors,tRNAlhe and tRNA he, were obtained from B. subtilis. tRNAIhe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA2he: in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N6-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU3-directed fornation of fMet-Phe, the undermodified tRNAphe is always less efficient at Mg2+ concentrations between 5 and 15 mM than its mature counterpart.

Iron mediated methylthiolation of tRNA as a regulator of operon expression inEscherichia coli

“…This tRNA also showed a marked reduction in its ability to bind to ribosomes in response to UAG. Undermodified E. coli tRNAphe lacking ms2 and other modifications, however, behaved normally in riboscne binding taining an unmodified G instead of Gm in the first position of the anticodon also supported peptide bond formation less efficiently than the fully modified tRNAphe (27). In view of these somewhat conflicting results we examined the ability of tRNA lacking ms from E. coli 0111 grown under iron restricted conditions to function in polypeptide synthesis.…”

Thionucleosides in transfer ribonucleic acid: diversity, structure, biosynthesis, and function.

“…E. coli trpX mutant exhibited an increased level of expression of all tryptophan operon enzymes due to reduced translational efficiency of tRNATTrp lacking the methylthioisopentenyl group, since structural gene expression through an attenuation mechanism is dependent upon the level of the translationally efficient charged tRNA present (33,123). There is considerable evidence that methylthiolation of i6A to ms2i6A in certain bacterial tRNAs is necessary for effective translation of messenger RNAs (55,74). A mutant of Saccharomyces cerevisiae isolated by its reduced ability to function as a tyrosine-inserting UAA nonsense suppressor contained very little isopentenyladenosine in the tRNA (66).…”

Deciphering the RNA Modification Landscape in Arabidopsis Chloroplast tRNAs and rRNAs Reveals a Blend of Ancestral and Acquired Characteristics

“…Methylation at G46 is a widespread modification across all life domains. It introduces a positive charge within the tertiary base pair with C13 and U22, thereby stabilising the 3D tRNA core (Figure 4a, b; (92)). Furthermore, its involvement in tRNA modification networks was documented.…”