Functional interaction between RNase III and the Escherichia coli ribosome

BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity. RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific. CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

BACKGROUND: The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. RESULTS: We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. CONCLUSION: Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes

Ribosome engineering reveals the importance of 5S rRNA autonomy for ribosome assembly

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis

Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity

Definition of bases in 23S rRNA essential for ribosomal subunit association

The ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. Recent structural studies of the 70S ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. We set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. Modification of the N-1 position of A715, A1912, or A1918 in Escherichia coli 50S subunits is strongly detrimental to 70S ribosome formation. This result points to 23S rRNA helices 34 and 69, and thus bridges B2a and B4, as essential for ensuring stability of the 70S ribosome

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly

Ribosome Protein Composition Mediates Translation during the E. coli Stationary Phase

Bacterial ribosomes contain over 50 ribosome core proteins (r-proteins). Tens of non-ribosomal proteins bind to ribosomes to promote various steps of translation or suppress protein synthesis during ribosome hibernation. This study sets out to determine how translation activity is regulated during the prolonged stationary phase. Here, we report the protein composition of ribosomes during the stationary phase. According to quantitative mass-spectrometry analysis, ribosome core proteins bL31B and bL36B are present during the late log and first days of the stationary phase and are replaced by corresponding A paralogs later in the prolonged stationary phase. Ribosome hibernation factors Rmf, Hpf, RaiA, and Sra are bound to the ribosomes during the onset and a few first days of the stationary phase when translation is strongly suppressed. In the prolonged stationary phase, a decrease in ribosome concentration is accompanied by an increase in translation and association of translation factors with simultaneous dissociation of ribosome hibernating factors. The dynamics of ribosome-associated proteins partially explain the changes in translation activity during the stationary phase