Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress

Abstract

Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.

Molecular mechanism of tRNA binding by theEscherichia coliN7 guanosine methyltransferase TrmB

“…This lack of growth for the BW25113 strain at high H2O2 concentrations has recently been corroborated by others (32). Given the unstable nature of hydrogen peroxide rendering it difficult to use precise concentrations and the conserved nature of this phenotype across different organisms, we conclude that trmB is important for fitness during oxidative stress.…”

RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA

“…In addition to Ψ, the retention of CMCT adduct after CMCT and alkaline treatment has been observed for 2methylthio-N6-isopentenyladenosine (ms 2 i 6 A) 38 and OH 5 C. 36 The positions of ms 2 i 6 A and OH 5 C modifications by CMCT remains unknown. 36,38 Here, we show that under our experimental conditions, OH 5 C 2505 and m 2 A 2507 retain the CMCT adducts after CMCT and NaHCO 3 treatment, and these adducts produce reverse transcriptase misincorporation and deletion (Figure 3A). The detection of OH 5 C 2505 and m 2 A 2507 using CMCT treatment was performed similar to Ψ detection experiments and as explained in the Materials and Methods section.…”

“…Furthermore, while there are more than 170 known RNA modifications, and many of these modifications have been implicated in antibiotic resistance, cancer, and neurodegenerative diseases, only a subset of RNA modifications can be detected in a high throughput and single nucleotide manner. ,,, The simultaneous detection of Ψ, m 3 Ψ, m 2 A, and OH 5 C modifications in a high throughput and in a single nucleotide resolution manner described in this study is readily generalizable to other RNA molecules. Lastly, MS, which is not a high throughput technique, has been used to detect OH 5 C on RNA. ,,,, Here, we have developed two techniques, involving CMCT and KMnO 4 treatments combined with misincorporations and deletion counting, to detect OH 5 C modification in RNA in a single nucleotide and high throughput manner.…”

“…Lastly, MS, which is not a high throughput technique, has been used to detect OH 5 C on RNA. 9,22,35,36,54 Here, we have developed two techniques, involving CMCT and KMnO 4 treatments combined with misincorporations and deletion counting, to detect OH 5 C modification in RNA in a single nucleotide and high throughput manner.…”

“…In addition, by treating 23S rRNA with KMnO 4 and counting the misincorporations and deletions of the reverse transcriptase, we are able to accurately detect OH 5 C. Therefore, employing our techniques, the OH 5 C could be identified in an RNA molecule using CMCT modification and validated using KMnO 4 modification. Historically OH 5 C has been detected in RNA using MS, 21,22,35,36 which is not a high throughput and single nucleotide resolution technique. Our techniques allow the detection of OH 5 C at a single nucleotide resolution and in a high throughput manner.…”