A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA

Abstract

Escherichia coli is the best-characterized organism with respect to posttranscriptional modifications of its ribosomal RNA (rRNA). It is presently believed that all the modified nucleotides have been identified, primarily on the basis of two detection methods; modification-induced inhibition of the enzyme reverse transcriptase or analysis by combined HPLC and electrospray ionization mass spectrometry. Comparison of data from these different approaches reveals a disagreement regarding modification of C2501 in E. coli 23S rRNA. A. Bakin and J. Ofengand previously reported the detection of a modification at this site based on a reverse transcriptase assay. J.A. McCloskey and coworkers could not confirm the existence of such a modification using an electrospray ionization mass spectrometry approach. C2501 is therefore generally considered unmodified. We have used a strategy involving isolation of a specific rRNA fragment from E. coli 23S rRNA followed by Matrix Assisted Laser Desorption/Ionization mass spectrometry and tandem mass spectrometry to investigate this controversy. Our data reveal a novel 16-Da partial modification at C2501. We believe that the data reported here clarify the above discrepancy, because a minor partial modification detected in a reverse transcriptase assay would not necessarily be detected by the original mass spectrometry approach. The level of modification was furthermore monitored in different growth situations, and we found a significant positive regulation in stationary phase cells. C2501 is universally conserved and implicated in structure folds very close to the catalytic center of the ribosome. Moreover, several antibiotics bind to nucleotides in this region, which altogether make a modification at this site interesting.

FIGURE 1.

Fragment C2480–C2527 of E. coli 23S rRNA digested with RNase T1. (A) MALDI time-of-flight mass spectrum of the m/z region covering trinucleotides or larger. The inset reveals the +16.0-Da partial modification of the 2496-CACmCUCG-2502 fragment at m/z 2249.27. (B) A list of the expected RNase T1 digestion fragments based on the rRNA sequence with presently known nucleotide modifications. m²A is 2-methyladenosine, and ψ indicates pseudouridine.

FIGURE 2.

Tandem mass spectra of the 2496-CACmCUCG-2502 fragment and its 16-Da-larger modified derivative (2215.35 and 2231.26 Da ions; 2′-3′-cyclic phosphate versions of the m/z 2233.32 and 2249.27 ions shown in Fig. 1A ▶). Lowercase letters refer to backbone cleavage fragments, where a, b, c, and d contain the original 5′-end, and w, x, y, and z the original 3′-end. Associated numbers are the length of the fragment in nucleotides. Capital letters refer to the nucleotides of internal fragments or, if preceded by (−) to loss of the indicated nucleobase.

FIGURE 3.

MALDI time-of-flight mass spectra of the region covering single nucleotides. (A) Fragment C2480–C2527 of E. coli 23S rRNA digested with the pyrimidine specific RNase A. Single cytidine and uridine monophosphates produced appear at the m/z values 324.05 and 325.05, respectively (the relative low intensity of the uridine monophosphate ion is caused by its lower proton affinity; Liguori et al. 2000). A peak corresponding to a 16-Da-modified cytidine monophosphate is furthermore observed at m/z 340.09. (B) The peaks of analyte ions are close to, but do not overlap, seven distinct matrix peaks in this region shown in the RNA-free control. (C) MALDI time-of-flight mass spectrum of a sample prepared as in A, but with an additional treatment with alkaline phosphatase. The peaks corresponding to cytidine, uridine, and 16-Da-modified cytidine are shifted to an 80-Da-lower value, corresponding to loss of phosphate.

FIGURE 4.

The m/z region 2190–2290 from an MALDI time-of-flight mass spectrum of the C2480–C2527 fragment of E. coli 23S rRNA digested with RNase T1. rRNA in this sample was isolated from stationary-phase cells. The 2496-CACmCUCG-2502 fragment at m/z 2233.34 and its 16-Da-larger derivative at 2249.31 appear at about equal intensities.