Dmitry E. Burakovsky

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation

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 m2G966 and m5C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m2G966/m5C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNAfMet to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m2G966 and m5C967 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.

Modifications of ribosomal RNA: From enzymes to function

Modified nucleosides are present in all kinds of stable RNA molecules, tRNAs being particularly rich in them (Auffinger and Westhof, 1998). Ribosomal RNA (rRNA) from all organisms contains modifications, and there is a correlation between the overall complexity of an organism and the number of modified nucleosides in its rRNA. The rRNA of the most primitive bacteria, such as some Mycoplasma species, may possess only 14 modified nucleosides (de Crecy-Lagard et al., 2007). In Escherichia coli, there are 36 modified nucleosides in rRNA (Table I). Yeast ribosomes possess about one hundred rRNA modifications, human rRNA over two hundred (Ofengand and Fournier, 1998; Decatur and Fournier, 2002). Eukaryotes and archaea use snoRNA guided rRNA modification mechanism. This mechanism allows archaea and eukarya to use a limited number of modification enzymes, mainly pseudouridine synthase and 2′-O-methyltransferase to introduce the majority of their rRNA modifications (Decatur and Fournier, 2002). By contrast, bacteria have developed specific enzymes for each one of the (fewer) modifications they have. Nevertheless, there are many different rRNA modifications in bacteria. Despite intensive study for several decades, many open questions remain regarding the functional role of modified rRNA nucleosides. In this review we will focus on rRNA modifications in E. coli and discuss their possible functions.

The structure of helix 89 of 23S rRNA is important for peptidyl transferase function of Escherichia coli ribosome

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.

Modified nucleotides m(2)G966/m(5)C967 of Escherichia coli 16S rRNA are required for attenuation of tryptophan operon.

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.

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction.

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.

Usage of rRNA-methyltransferase for site-specific fluorescent labeling

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.

The interaction with Escherichia coli 23S rRNA helices 89 and 91 contributes to the IF2 activity but is insignificant for the functioning of the elongation factors

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.