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We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

Previously, we showed that strains which have been deleted for the 21K gene (hereafter called yfjA), of the trmD operon, encoding a 21-kDa protein (21K protein) have an approximately fivefold-reduced growth rate in rich medium. Here we show that such mutants show an up to sevenfold reduced growth rate in minimal medium, a twofold-lower cell yield-to-carbon source concentration ratio, and a reduced polypeptide chain growth rate of ␤-galactosidase. Suppressor mutations that increased the growth rate and translational efficiency of a ⌬yfjA mutant were localized to the 3 part of rpsM, encoding ribosomal protein S13. The 21K protein was shown to have affinity for free 30S ribosomal subunits but not for 70S ribosomes. Further, the 21K protein seems to contain a KH domain and a KOW motif, both suggested to be involved in binding of RNA. These findings suggest that the 21K protein is essential for a proper function of the ribosome and is involved in the maturation of the ribosomal 30S subunits or in translation initiation.A majority of the ribosomal protein (r-protein) genes in Escherichia coli are located in the region between 72 and 73 min on the genetic map, whereas the remaining genes are scattered around the chromosome in smaller operons of one to four genes (1). In addition to r-protein genes, many of the r-protein operons contain genes for nonribosomal proteins. For example, the genes for elongation factors G and Tu are found in the str operon, whereas the ␣ operon (rpsM-rpsKrpsD-rpoA-rplQ), the macromolecular synthesis operon (rpsUdnaG-rpoD), and the rplKAJL-rpoBC operon encode the subunits of the RNA polymerase (see reference 23). There is no obvious reason why nonribosomal protein genes are located within r-protein operons; however, it has been speculated that during certain conditions there may be a need for a coordinated synthesis of the ribosomal and nonribosomal proteins due to functional relationships (3).The trmD operon contains the genes for r-proteins S16 (rpsP) and L19 (rplS), the gene for the tRNA (m 1 G37)methyltransferase (trmD), and the 21K gene (hereafter called yfjA), encoding a 21-kDa protein called the 21K protein (10). As part of a functional analysis of the E. coli genome, the genes in the trmD operon were subjected to allelic replacement by inactivated gene copies (38). R-proteins S16 and L19 were both found to be essential for viability. Moreover, a strain carrying a slightly polar insertion upstream from the rplS gene was defective in the assembly of the 50S ribosomal subunits due to limiting amounts of r-protein L19. In vitro, r-protein S16 has been shown to be essential for efficient assembly of the 30S ribosomal subunits (21) in accordance with its essentiality in vivo (38). Thus, both S16 and L19 are important for assembly of the ribosomal subunits. The tRNA (m 1 G37)methyltransferase modifies the guanosine in position 37 next to the anticodon of a subset of the tRNAs in E. coli and Salmonella typhimurium (4, 5). Studies on an S. typhimurium trmD mutant demonstrated that the modified ...