Nucleotide sequence of the ksgA gene of Escherichia coli: comparison of methyltransferases effecting dimethylation of adenosine in ribosomal RNA.

The ksgA gene of Escherichia coli encodes a methyltransferase (MeT) that specifically dimethylates two adjacent adenosines near the 3' end of 16S RNA in the 30S particle. Its inactivation leads to kasugamycin (Ksg) resistance. Several plasmids were constructed with inserts which complemented chromosomal ksgA mutations. One of these inserts was sequenced and found to contain an open reading frame (ORF) sufficient to code for the previously identified 30-kDal MeT. We have compared the amino acid (aa) sequence of the ksgA-encoded enzyme with three published sequences of MeT involved in dimethylation of an adenosine residue in 23S RNA and rendering the organisms resistant to the MLS antibiotics. The homologous patches in the sequences of all four enzymes suggest that those might correspond to contact points for the common substrates, e.g., for the adenosine residue(s) and S-adenosylmethionine (SAM).

Partial methylation of two adjacent adenosines in ribosomes from Euglena gracilis chloroplasts suggests evolutionary loss of an intermediate stage in the methyl-transfer reaction.

Bacterial, cytoplasmic and organellar ribosomes from a wide phylogenetic spectrum of organisms have a characteristic m6(2)Am6(2)A structure near the 3' end of the RNA of the small ribosomal subunit (SSU). We have studied one of the few exceptions to this extremely conserved post-transcriptionally modified sequence, i.e. dimethylation of only one of the two A's in chloroplasts from Euglena gracilis. It was established that only the A closest to the 5' end is dimethylated, the other one being unmodified. The methylation reaction was studied in vitro using ribosomes from a kasugamycin resistant mutant (ksgA) of Escherichia coli and purified methyl-transferase. Using limited amounts of the methyl donor S-adenosylmethionine (SAM) a partial level of methylation (50% of control) was attained. It is shown that in this case the 3' proximal A is dimethylated while the other is not. This suggests that dimethylation takes place in two successive stages. Apparently in E. gracilis chloroplasts the first stage of methylation does not occur.

Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene.

The aminoglycoside kasugamycin, which has previously been shown to inhibit initiation of protein biosynthesis in vitro, also affects translational accuracy in vitro. This is deduced from the observation that the drug decreases the incorporation of histidine relative to alanine into the coat protein of phage MS2, the gene of which is devoid of histidine codons. The read-through of the MS2 coat cistron, due to frameshifts in vitro, is also suppressed by the antibiotic. In contrast, streptomycin enhances histidine incorporation and read-through in this system. The effects of kasugamycin take place at concentrations that do not inhibit coat protein biosynthesis. Kasugamycin-resistant mutants (ksgA) lacking dimethylation of two adjacent adenosines in 16 S ribosomal RNA, show an increased leakiness of nonsense and frameshift mutants (in the absence of antibiotic). They are therefore phenotypically similar to previously described ribosomal ambiguity mutants (ram).

Kasugamycin resistant mutants of Bacillus stearothermophilus lacking the enzyme for the methylation of two adjacent adenosines in 16S ribosomal RNA.

Several mutants of B. stearothermophilus have been isolated that are resistant to the antibiotic kasugamycin. One of these is shown to lack dimethylation of two adjacent adenosines in the 16S ribosomal RNA. All mutants that were analyzed biochemically lack the enzyme that is able to methylate this site. Ribosomal sensitivity and resistance to kasugamycin in B. stearothermophilus is therefore, like in E. coli, closely connected with dimethylation of the adenosines.

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16 S ribosomal RNA of Escherichia coli. II. The effect of the absence of the methyl groups on initiation of protein biosynthesis.

The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine.