Ribosomes contain a number of modifications in rRNA, the function of which is unclear. Here we show – using proteomic analysis and dual fluorescence reporter in vivo assays – that m2G966 and m5C967 in 16S rRNA of Escherichia coli ribosomes are necessary for correct attenuation of tryptophan (trp) operon. Expression of trp operon is upregulated in the strain where RsmD and RsmB methyltransferases were deleted, which results in the lack of m2G966 and m5C967 modifications. The upregulation requires the trpL attenuator, but is independent of the promotor of trp operon, ribosome binding site of the trpE gene, which follows trp attenuator and even Trp codons in the trpL sequence. Suboptimal translation initiation efficiency in the rsmB/rsmD knockout strain is likely to cause a delay in translation relative to transcription which causes misregulation of attenuation control of trp operon.

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m(2)G966 and m(5)C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m(2)G966/m(5)C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNA(fMet) to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m(2)G966 and m(5)C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.

The possibility of using an S-adenosylmethionine analog, i.e., pent-2-en-4-ynyl S-adenosylhomocysteine (AduEnYn), as an rRNA methyltransferase cofactor has been investigated. The conditions for the cycloaddition reaction of the fluorescent label to the S-adenosylmethionine analog were chosen. The functional activity of E. coli ribosomes was tested under different conditions. It was found that the introduction of the alkynyl radical occurred successfully and did not affect the functional activity of the ribosome; however, the inactivation of the ribosome occurred during the following cycloaddition reaction.

Helix 89 of the 23S rRNA connects ribosomal peptidyltransferase center and elongation factor binding site. Secondary structure of helix 89 determined by X-ray structural analysis involves less base pairs then could be drawn for the helix of the same primary structure. It can be that alternative secondary structure might be realized at some stage of translation. Here by means of site-directed mutagenesis we stabilized either the "X-ray" structure or the structure with largest number of paired nucleotides. Mutation UU2492-3C which aimed to provide maximal pairing of the helix 89 of the 23S rRNA was lethal. Mutant ribosomes were unable to catalyze peptide transfer independently either with aminoacyl-tRNA or puromycin.

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a "gate" at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

The noncanonical pairing of C2475 with G2529 links 23S rRNA helices 89 and 91 in the Escherichia coli ribosome. These nucleotides are at the intersection of the peptidyltransferase center, the sarcin-ricin loop, and the GTPase-associated center of the ribosome. The functional significance of C2475 and G2529 was studied using the C2475G, C2475G/G2529C, and ΔA2471/U2479 mutations of the 23S rRNA. The mutations did not change the activity of the elongation factors, but affected the cell growth rate, the 23S rRNA conformation, and the translation initiation. The C2475G and C2475G/G2529C mutations substantially affected the binding of the IF2 · GDPNP complex to the ribosome and the IF2-dependent formation of the initiation complex and increased the ribosome-stimulated GTPase activity of IF2. The Δ A2471/U2479 mutation did not affect the binding of IF2 · GDPNP to the ribosome, but influenced the IF2-dependent formation of the initiation complex and GTPase activity of IF2. The contact between helices 89 and 91 was found to be important for the efficient translation initiation catalyzed by IF2.

The noncanonical pairing of C2475 with G2529 links 23S rRNA helices 89 and 91 in the Escherichia coli ribosome. These nucleotides are at the intersection of the peptidyltransferase center, the sarcin–ricin loop, and the GTPase-associated center of the ribosome. The functional significance of C2475 and G2529 was studied using the C2475G, C2475G/G2529C, and ∆ A 2471/ U 2479 mutations of the 23S rRNA. The mutations did not change the activity of the elongation factors, but affected the cell growth rate, the 23S rRNA conformation, and the translation initiation. The C2475G and C2475G/G2529C mutations substantially affected the binding of the IF2 · GDPNP complex to the ribosome and the IF2-dependent formation of the initiation complex and increased the ribosome-stimulated GTPase activity of IF2. The ∆ A2471/U2479 mutation did not affect the binding of IF2 · GDPNP to the ribosome, but influenced the IF2-dependent formation of the initiation complex and GTPase activity of IF2. The contact between helices 89 and 91 was found to be important for the efficient translation initiation catalyzed by IF2. DOI: 10.1134/S0026893307060118