Identification of the rrmA gene encoding the 23S rRNA m1G745 methyltransferase in Escherichia coli and characterization of an m1G745-deficient mutant

Abstract

An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83-100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.

FIG. 1

The modified nucleotide m¹G is lacking in 23S rRNA from strain IB103. HPLC chromatograms of nucleosides from the 50S subunits of strain CP79 (wild type) and strain IB103 (rrmA10) are shown. The HPLC chromatogram of RNA from strain IB10 is identical to that of IB103 (data not shown). Peak 1 corresponds to the modified nucleoside Gm, peak 2 corresponds to m¹G, and peak 3 corresponds to m²G. AU, absorption units.

FIG. 2

The rrmA-dependent m¹G modification is located at position 745 of 23S rRNA. The autoradiogram shows reverse transcription of 23S rRNA from strains CP79 (wild type), IB10, and IB103 (both lacking m¹G745). Also included is RNA sequencing with the same primer as that used for reverse transcription.

FIG. 3

Sucrose gradient sedimentation profiles of extracts from strains CP79 (A) and IB103 (B) made under nonstringent conditions (10 mM Mg²⁺). The positions of tRNA, 30S and 50S ribosomal subunits, 70S monosomes, and polysomes are indicated.

FIG. 4

Gene organization of the chromosomal inserts of plasmids pSJ6 (21) and pBP51. Both plasmids are derivatives of pUC19 (39). Designations of ORFs are from reference , where o and f represent different orientations of transcription and the number gives the length of the gene product in amino acids.

FIG. 5

Viomycin protects G914 100-fold better in strains lacking m¹G745. Shown is protection by viomycin against kethoxal modification at base G914 in ribosomes containing or lacking m¹G745. The autoradiogram band intensities were measured with a PhosphorImager and are given as the ratio to the protection of C908, a base which is unaffected by viomycin and kethoxal. Each bar represents at least two independent experiments; in each experiment, the samples were done in triplicate. ▪, strain IB103 (rrmA); □, strain CP79 (wild type).

FIG. 6

The modified nucleotide m¹G is present in purple bacteria. The autoradiogram shows reverse transcription of 23S from various bacterial sources as noted. The position of G745 or its equivalent is marked.