RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives.

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

Cooperative nature of gating transitions in K+ channels as seen from dynamic importance sampling calculations

The growing dataset of K⁺ channel x‐ray structures provides an excellent opportunity to begin a detailed molecular understanding of voltage‐dependent gating. These structures, while differing in sequence, represent either a stable open or closed state. However, an understanding of the molecular details of gating will require models for the transitions and experimentally testable predictions for the gating transition. To explore these ideas, we apply dynamic importance sampling to a set of homology models for the molecular conformations of K⁺ channels for four different sets of sequences and eight different states. In our results, we highlight the importance of particular residues upstream from the Pro‐Val‐Pro (PVP) region to the gating transition. This supports growing evidence that the PVP region is important for influencing the flexibility of the S6 helix and thus the opening of the gating domain. The results further suggest how gating on the molecular level depends on intra‐subunit motions to influence the cooperative behavior of all four subunits of the K⁺ channel. We hypothesize that the gating process occurs in steps: first sidechain movement, then inter‐S5‐S6 subunit motions, and lastly the large‐scale domain rearrangements. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit

Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we characterized, both compositionally and structurally, a set of in vivo 50S subunit precursors (45S), isolated from a mutant bacterial strain. Our qualitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are dramatically underrepresented in the 45S particles. This protein spectrum shows interesting similarity to many qualitatively analyzed 50S precursors from different genetic background, indicating the presence of global rate-limiting steps in the late-stage assembly of 50S subunit. Our structural data reveal two major intermediate states for the 45S particles. Consistently, both states severally lack those proteins, but they also differ in the stability of the functional centers of the 50S subunit, demonstrating that they are translationally inactive. Detailed analysis indicates that the orientation of H38 accounts for the global conformational differences in these intermediate structures, and suggests that the reorientation of H38 to its native position is rate-limiting during the late-stage assembly. Especially, H38 plays an essential role in stabilizing the central protuberance, through the interaction with the 5S rRNA, and the correctly orientated H38 is likely a prerequisite for further maturation of the 50S subunit.     

Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer

We have used F?rster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 S subunits and 70 S ribosomes from X-ray and cryo-electron microscope structures, and the platform is predicted to undergo a clock-wise rotation upon association.     

Modeling subunit cooperativity in opening of tetrameric ion channels

Most potassium channels are tetramers of four homologous polypeptides (subunits). During channel gating, each subunit undergoes several conformational changes independent of the state of other subunits before reaching a permissive state, from which the channel can open. However, transition from the permissive states to the open state involves a concerted movement of all subunits. This cooperative transition must be included in Markov models of channel gating. Previously, it was implemented by considering all possible combinations of four subunit states in a much larger expanded model of channel states (e.g., 27,405 channel states versus 64 subunit states), which complicates modeling and is computationally intense, especially when accurate modeling requires a large number of subunit states. To overcome these complexities and retain the tetrameric molecular structure, a modeling approach was developed to incorporate the cooperative transition directly from the subunit models. In this approach, the open state is separated from the subunit models and represented by the net flux between the open state and the permissive states. Dynamic variations of the probability of state residencies computed using this direct approach and the expanded model were identical. Implementation of the direct approach is simple and its computational time is orders-of-magnitude shorter than the equivalent expanded model.     

Savoryellales (Hypocreomycetidae, Sordariomycetes): a novel lineage of aquatic ascomycetes inferred from multiple-gene phylogenies of the genera Ascotaiwania, Ascothailandia, and Savoryella

The taxonomic placement of freshwater and marine Savoryella species has been widely debated, and the genus has been tentatively assigned to various orders in the Sordariomycetes. The genus is characterized as possessing paraphyses that deliquesce early, elongate, clavate to cylindrical asci with a poorly developed apical ring and versicolored, three-septate ascospores. We performed two combined phylogenetic analyses of different genes: (i) partial small subunit rRNA (SSU), large subunit rRNA (LSU), DNA-dependent RNA polymerase II largest subunit (rpb2) dataset and (ii) SSU rDNA, LSU rDNA, DNA-dependent RNA polymerase II largest subunit (rpb1 and rpb2), translation elongation factor 1-alpha (tef1), the 5.8S ribosomal DNA (5.8S rDNA) dataset. Our results indicate that Savoryella species formed a monophyletic group within the Sordariomycetes but showed no affinity to the Hypocreales, Halosphaeriales (now Microascales), Sordariales and Xylariales, despite earlier assignments to these orders. Savoryella, Ascotaiwania and Ascothailandia (and its anamorph, Canalisporium) formed a new lineage that has invaded both marine and freshwater habitats, indicating that these genera share a common ancestor and are closely related. Because they show no clear relationship with any named order we erect a new order Savoryellales in the subclass Hypocreomycetidae, Sordariomycetes. The genera Savoryella and Ascothailandia are monophyletic, while the position of Ascotaiwania is unresolved. All three genera are phylogenetically related and form a distinct clade similar to the unclassified group of marine ascomycetes comprising the genera Swampomyces, Torpedospora and Juncigera (TBM clade: Torpedospora/Bertia/Melanospora) in the Hypocreomycetidae incertae sedis.     

Molecular systematics of sponges (Porifera)

The first application of molecular systematics to sponges was in the 1980s, using allozyme divergence to dis-criminate between conspecific and congeneric sponge populations. Since this time, a fairly large database has been accumulated and, although the first findings seemed to indicate that sponge species were genetically more divergent than those of other marine invertebrates, a recent review of the available dataset indicates that levels of interspecific gene identities in most sponges fall within the normal range found between species of other invertebrates. Nevertheless, some sponge genera have species that are extremely divergent from each other, suggesting a possible polyphyly of these genera. In the 1990s, molecular studies comparing sequences of ribosomal RNA have been used to reappraise the phylogenetic relationships among sponge genera, families, orders and classes. Both the 18S small subunit and the 28S large subunit rRNA genes have been sequenced (41 complete or partial and 75 partial sequences, respectively). Sequences of 18S rRNA show good support for Porifera being true Metazoa, but they are not informative for resolving relationships among genera, families or orders. 28S rRNA domains D1 and D2 appear to be more informative for the terminal nodes and provide resolution for internal topologies in sufficiently closely related species, but the deep nodes between orders or classes cannot be resolved using this molecule. Recently, a more conserved gene, Hsp70, has been used to try to resolve the relationships in the deep nodes. Metazoan monophyly is very well supported. Nevertheless, the divergence between the three classes of Porifera, as well as the divergence between Porifera, Cnidaria and Ctenophora, is not resolved. Research is in progress using other genes such as those of the homeodomain, the tyrosine kinase domain, and those coding for the aggregation factor. For the moment the dataset for these genes is too restricted to resolve the phylogenetic relationships of these phyla. However, whichever the genes, the phylogenies obtained suggest that Porifera could be paraphyletic and that the phylogenetic relationships of most of the families and orders of the Demospongiae have to be reassessed. The Calcarea and Hexactinellida are still to be studied at the molecular level.     

Orientations and Proximities of the Extracellular Ends of Transmembrane Helices S0 and S4 in Open and Closed BK Potassium Channels

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V₅₀ for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.     

RNA conformational classes

RNA exhibits a large diversity of conformations. Three thousand nucleotides of 23S and 5S ribosomal RNA from a structure of the large ribosomal subunit were analyzed in order to classify their conformations. Fourier averaging of the six 3D distributions of torsion angles and analyses of the resulting pseudo electron maps, followed by clustering of the preferred combinations of torsion angles were performed on this dataset. Eighteen non-A-type conformations and 14 A-RNA related conformations were discovered and their torsion angles were determined; their Cartesian coordinates are available.     

Structural insights into the function of a unique tandem GTPase EngA in bacterial ribosome assembly

Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria, and intriguingly, it contains two consecutive GTPase domains (GD), being one-of-a-kind among all known GTPases. EngA is required for the 50S subunit maturation. However, its molecular role remains elusive. Here, we present the structure of EngA bound to the 50S subunit. Our data show that EngA binds to the peptidyl transferase center (PTC) and induces dramatic conformational changes on the 50S subunit, which virtually returns the 50S subunit to a state similar to that of the late-stage 50S assembly intermediates. Very interestingly, our data show that the two GDs exhibit a pseudo-two-fold symmetry in the 50S-bound conformation. Our results indicate that EngA recognizes certain forms of the 50S assembly intermediates, and likely facilitates the conformational maturation of the PTC of the 23S rRNA in a direct manner. Furthermore, in a broad context, our data also suggest that EngA might be a sensor of the cellular GTP/GDP ratio, endowed with multiple conformational states, in response to fluctuations in cellular nucleotide pool, to facilitate and regulate ribosome assembly.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.     

Hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16S rRNA in the 30S ribosome

Background  

Conformational flexibility in structured RNA frequently is critical to function. The 30S ribosomal subunit exists in different conformations in different functional states due to changes in the central part of the 16S rRNA. We are interested in evaluating the factors that might be responsible for restricting flexibility to specific parts of the 16S rRNA using biochemical data obtained from the 30S subunit in solution. This problem was approached taking advantage of the observation that there must be a high degree of conformational flexibility at sites where UV photocrosslinking occurs and a lack of flexibility inhibits photoreactivity at many other sites that are otherwise suitable for reaction.     

Phylogenetic analyses of the rhipicephaline ticks indicate that the genus Rhipicephalus is paraphyletic

We inferred the phylogeny of 21 species and subspecies of ticks from the subfamilies Rhipicephalinae and Hyalomminae using cytochrome c oxidase subunit I (COI) and 12S rRNA mitochondrial gene sequences. Two members of the subfamily Haemaphysalinae were used for outgroup reference. The largest rhipicephaline genus, Rhipicephalus, was represented by ticks from six of the species groups, the second largest genus, Dermacentor, by species from two of three of its subgenera, and the genus Boophilus by 3 of its 5 species. We analyzed the 12S and COI sequences separately and together; statistically significant incongruence between the 12S rDNA and the COI sequences was not detected in the combined dataset using the incongruence length difference test. The combined dataset provided greater phylogenetic resolution than the individual datasets, and although the 12S rDNA data had only 25% of the parsimony-informative characters, it provided half of the total partitioned Bremer support for the combined dataset. We present the first hypothesis of phylogenetic relationships among some species groups of Rhipicephalus but our most controversial result was that the genus Rhipicephalus is apparently paraphyletic, unless species of Boophilus are included in it. The species of Rhipicephalus most closely related to Boophilus spp. were from the R. pravus and R. evertsi species groups, which may implicate an African origin for this important group of ticks.     

Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements

Initiation factors, elongation factors, and release factors all interact with the L7/L12 stalk of the large ribosomal subunit during their respective GTP-dependent cycles on the ribosome. Electron density corresponding to the stalk is not present in previous crystal structures of either 50 S subunits or 70 S ribosomes. We have now discovered conditions that result in a more ordered factor-binding center in the Haloarcula marismortui (H.ma) large ribosomal subunit crystals and consequently allows the visualization of the full-length L11, the N-terminal domain (NTD) of L10 and helices 43 and 44 of 23 S rRNA. The resulting model is currently the most complete reported structure of a L7/L12 stalk in the context of a ribosome. This region contains a series of intermolecular interfaces that are smaller than those typically seen in other ribonucleoprotein interactions within the 50 S subunit. Comparisons of the L11 NTD position between the current structure, which is has an NTD splayed out with respect to previous structures, and other structures of ribosomes in different functional states demonstrates a dynamic range of L11 NTD movements. We propose that the L11 NTD moves through three different relative positions during the translational cycle: apo-ribosome, factor-bound pre-GTP hydrolysis and post-GTP hydrolysis. These positions outline a pathway for L11 NTD movements that are dependent on the specific nucleotide state of the bound ligand. These three states are represented by the orientations of the L11 NTD relative to the ribosome and suggest that L11 may play a more specialized role in the factor binding cycle than previously appreciated.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.     

Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

Deprotonation of the 33-kDa, extrinsic, manganese-stabilizing subunit accompanies photooxidation of manganese in photosystem II.

Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform (13)C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S(1) to S(2) transition in the presence either of reconstituted (12)C or (13)C MSP. (13)C labeling of MSP is shown to cause 30-60 cm(-1) shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/aspartic acid residues on MSP during the S(1) to S(2) transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.