Studies on the culture condition dependent changes of modified nucleosides in the transfer RNA of azotobacter vinelandii

Abstract

Transfer RNA (tRNA), the key molecule involved in the translation of genetic information, contains a variety of modified nucleosides at specific positions in the molecule. These modified nucleosides are formed by post?transcriptional modifications of adenosine, cytidine, guanosine, or uridine, and have been shown to play crucial roles in the different functions of tRNA. The different kinds of modifications found in tRNAs include methylation, thiolation, and the introduction of groups such as an isopentenyl group, carboxymethylaminomethyl group, threonine, glycine, and others. These modifications influence key aspects of tRNA biology, including stabilization of the anticodon loop and optimization of codon–anticodon interactions during protein synthesis. It is well known that organisms adapt to wide fluctuations in temperature, nutrient availability, or the presence of growth inhibitors in their natural habitat by regulating cellular metabolism. In recent years, it has become increasingly clear that organisms regulate their metabolism in response to environmental influences by altering the modifications of the nucleosides in tRNA, thereby modulating the functions of the tRNA molecule. For instance, Escherichia coli tRNAs normally contain the modified base 2?methylthio?N??isopentenyladenosine (ms²i?A), but under certain growth conditions these tRNAs can contain isopentenyladenosine (i?A), leading to reduced translational efficiency because of under?modification. This reduction in translational efficiency can relieve transcription termination at certain operons, thereby enhancing gene expression during stress. In bacteria such as Azotobacter vinelandii, which can fix atmospheric nitrogen in free?living conditions and also grow in the presence of excess ammonium, the metabolic status of the cell differs significantly between nitrogen?fixing and non?fixed growth conditions. The present study thus attempts to analyze the influence of these differences in metabolic status on the level of modifications found in tRNA from A. vinelandii. Total tRNA was isolated from cells grown under nitrogen?fixing (ammonium?free) and non?nitrogen?fixing (excess ammonium) conditions, hydrolyzed to mononucleotides or mononucleosides, and analyzed by electrophoretic mobility, thin?layer chromatography, column chromatography, and spectral measurements. Four prominent thiolated nucleosides were found in total tRNA from cells grown without ammonium: 5?methylaminomethyl?2?thiouridine (mnm?s²U), 2?methylthioisopentenyladenosine (ms²i?A), 2?methylthio?ribosylzeatin (ms²oA), and 4?thiouridine (s?U). The relative proportion of s?U was the highest, accounting for 45?% of the total sulfur incorporated, with other thiolated nucleosides present in smaller proportions. In contrast, tRNA from cells grown with excess ammonium showed dramatic changes in the thiolated nucleoside pattern. The proportion of s?U decreased from 45?% to 5?%, and a new thiolated species, comprising about 29?% of labeled sulfur, appeared. Meanwhile, the relative proportion of mnm?s²U decreased to 12?%, and both ms²i?A and ms²oA increased to about 29?% each. This indicates that growth conditions significantly affect the patterns of tRNA nucleoside modification, likely reflecting changes in the metabolic status of the cell.

Additionally, total tRNA from ammonium?grown cells lacked ribothymidine (T), a universally conserved nucleoside normally found at position 54 of the tRNA T?loop and involved in stable codon–anticodon interactions during translation, further demonstrating the impact of environmental conditions on tRNA modification.