Cold shock and its effect on ribosomes and thermal tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0 degrees C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 +/- 0.1 degrees C (mean +/- standard deviation) to 72.1 +/- 0.5 degrees C (P < or = 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 +/- 0.4 degrees C to 66.8 +/- 0.5 degrees C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states

HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.     

RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA

Pseudouridine modifications in helix 69 (H69) of 23S ribosomal RNA are highly conserved among all organisms. H69 associates with helix 44 of 16S rRNA to form bridge B2a, which plays a vital role in bridging the two ribosomal subunits and stabilizing the ribosome. The three pseudouridines in H69 were shown earlier to play an important role in 50S subunit assembly and in its association with the 30S subunit. In Escherichia coli, these three modifications are made by the pseudouridine synthase, RluD. Previous work showed that RluD is required for normal ribosomal assembly and function, and that it is the only pseudouridine synthase required for normal growth in E. coli. Here, we show that RluD is far more efficient in modifying H69 in structured 50S subunits, compared to free or synthetic 23S rRNA. Based on this observation, we suggest that pseudouridine modifications in H69 are made late in the assembly of 23S rRNA into mature 50S subunits. This is the first reported observation of a pseudouridine synthase being able to modify a highly structured ribonucleoprotein particle, and it may be an important late step in the maturation of 50S ribosomal subunits.     

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.     

Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Species-specific antibiotic-ribosome interactions: implications for drug development

In the cell, the protein synthetic machinery is a highly complex apparatus that offers many potential sites for functional interference and therefore represents a major target for antibiotics. The recent plethora of crystal structures of ribosomal subunits in complex with various antibiotics has provided unparalleled insight into their mode of interaction and inhibition. However, differences in the conformation, orientation and position of some of these drugs bound to ribosomal subunits of Deinococcus radiodurans (D50S) compared to Haloarcula marismortui (H50S) have raised questions regarding the species specificity of binding. Revisiting the structural data for the bacterial D50S-antibiotic complexes reveals that the mode of binding of the macrolides, ketolides, streptogramins and lincosamides is generally similar to that observed in the archaeal H50S structures. However, small discrepancies are observed, predominantly resulting from species-specific differences in the ribosomal proteins and rRNA constituting the drug-binding sites. Understanding how these small alterations at the binding site influence interaction with the drug will be essential for rational design of more potent inhibitors.     

The central core region of yeast ribosomal protein L11 is important for subunit joining and translational fidelity

Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunit-joining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented. These findings may have implications for our understanding of multifaceted diseases such as Diamond–Blackfan anemia which have been linked in part with mutations in L11.     

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation.     

Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

Multiple e-Pharmacophore modeling to identify a single molecule that could target both streptomycin and paromomycin binding sites for 30S ribosomal subunit inhibition

The bacterial ribosome is an established target for anti-bacterial therapy since decades. Several inhibitors have already been developed targeting both defined subunits (50S and 30S) of the ribosome. Aminoglycosides and tetracyclines are two classes of antibiotics that bind to the 30S ribosomal subunit. These inhibitors can target multiple active sites on ribosome that have a complex structure. To screen putative inhibitors against 30S subunit of the ribosome, the crystal structures in complex with various known inhibitors were analyzed using pharmacophore modeling approach. Multiple active sites were considered for building energy-based three-dimensional (3D) pharmacophore models. The generated models were validated using enrichment factor on decoy data-set. Virtual screening was performed using the developed 3D pharmacophore models and molecular interaction towards the 30S ribosomal unit was analyzed using the hits obtained for each pharmacophore model. The hits that were common to both streptomycin and paromomycin binding sites were identified. Further, to predict the activity of these hits a robust 2D-QSAR model with good predictive ability was developed using 16 streptomycin analogs. Hence, the developed models were able to identify novel inhibitors that are capable of binding to multiple active sites present on 30S ribosomal subunit.     

Cold Shock and Its Effect on Ribosomes and Thermal Tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0°C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 ± 0.1°C (mean ± standard deviation) to 72.1 ± 0.5°C (P ≤ 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 ± 0.4°C to 66.8 ± 0.5°C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation

Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.     

Isolation and characterization of a dominant negative mutant of Bacillus subtilis GTP-binding protein, YlqF, essential for biogenesis and maintenance of the 50 S ribosomal subunit

The circularly permuted GTPase YlqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of YlqF (YlqFDeltaN10) inhibits cell growth even in the presence of wild-type YlqF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqFDeltaN10 acts as a competitive inhibitor of wild-type YlqF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqFDeltaN10-overexpressing cells and in YlqF-depleted cells, suggesting that YlqFDeltaN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFDeltaN10-overexpressing and YlqF-depleted cells more strongly enhanced the GTPase activity of YlqF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that YlqF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.