U modifications have previously been identified in E.coli:
one at tRNA position 54, and two in 23S rRNA at positions 747 and 1939 (2
). In this study we have used MALDI mass spectrometry to link the three sites of m5
U modification with their respective methyltransferase enzymes. The tRNA m5
U54 methyltransferase TrmA is known to retain its activity after purification (30
), and the enzyme served as a positive control for our in vitro
methylation assays. A search of the E.coli
genome at the inception of our studies confirmed that the two most likely m5
U rRNA methyltransferase candidates were YgcA and YbjF.
Recombinant YgcA proved to be as easy to purify and handle as TrmA, retaining its activity and specificity in vitro.
YgcA specifically methylated an RNA transcript of 66 nucleotides, which was designed to mimic the structure around 23S rRNA nucleotide U1939 (Fig. C). Analysis of the 66mer after RNase digestion showed that YgcA increased the mass of the sequence 1936–1945 by 14 Da in the presence of the methyl group donor, SAM. This is consistent with the addition of a single methyl group at U1939 by YgcA. During the course of our study, Agarwalla and co-workers pinpointed the YgcA target at U1939 using biochemical approaches; they subsequently renamed this methyltransferase RumA (RNA uridine methyltransferase A) (21
By a process of elimination, this left YbjF as the most likely candidate to target the U747 site. However, despite the construction of a variety of recombinant versions of YbjF and the use of different enzyme purification methods and RNA substrates, the study of YbjF function in vitro proved to be intractable. A second approach was taken, which involved analysis of authentic rRNA. The large subunit rRNAs (5S and 23S rRNAs) give rise to over 900 fragments upon RNase T1 digestion, and analysis of these required stretching the limits of the MALDI technology. Fortuitously, the putative target of YbjF occurs within the RNase T1 digestion product 5′-ACUAAUm1Gψm5UGp (nucleotides 739–748), which is a double fragment formed by virtue of the RNase-resistant m1G745 nucleotide. This fragment forms a distinct peak in the mass spectrum (Fig. C), the position of which is shifted 14 Da downstream corresponding to the loss of a single methyl group in the strain without a functional YbjF protein (Fig. D). The position of the methyl group added by YbjF could be localised within reasonable doubt to U747 by using strains that lack the m1G745 modification.
Most rRNA modifications are located within preserved, functionally important regions of the rRNA (6
). The YgcA (RumA) target U1939 is phylogenetically conserved, with a uridine at this position in >95% of all organisms (Gutell Lab Comparative RNA: http://www.rna.icmb.utexas.edu
). Similarly, YbjF (now RumB) targets a nucleotide that is conserved as a uridine in >95% of all bacteria, and in >90% of organisms in the other two phylogenetic domains (data from the Gutell web site).
Other enterobacteria possess homologues of rumA
, and homologues are also evident in less closely related Gram-negative bacteria, as well as in phylogenetically distant species of archaea (Table ). The conserved, methylated uridines are situated in regions of the 23S rRNA that have been associated with essential functions in protein synthesis. U1939 is located within the rRNA structure that interacts with the aminoacyl end of A-site bound tRNA (4
), and could play an active role in sensing uncharged tRNA (21
). U747 is situated in a highly conserved and densely modified part of the rRNA that lines a constricted region of the 50S subunit tunnel through which the nascent peptide chain passes during protein synthesis (3
). This region of the tunnel additionally forms a binding pocket for macrolides and other antibiotics (39
). Structural changes here confer antibiotic resistance (47
), and can perturb regulatory interactions with the nascent peptide chain (49
). Therefore, given its conservation and ribosomal location, the U747 methylation might be expected to be involved in one of more of these processes. However, in competition experiments run in rich and minimal media, we have not been able to detect any growth disadvantage for ybjF
) knockout strains compared to the wild type (not shown). The presence of the U747 methylation could become important under specific growth or stress conditions, but definition of such a physiological role presently remains elusive.
Homologues of YgcA(RumA) and YbjF(RumB)