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Magnesium (Mg 2+ ) is the most abundant divalent cation in the cell and is critical for numerous cellular processes. Despite its importance, the mechanisms of intracellular Mg 2+ transport and its regulation are poorly understood. MgtE is the main Mg 2+ transport system in almost half of bacterial species and is an ortholog of mammalian SLC41A1 transporters, which are implicated in neurodegenerative diseases and cancer. To date, only MgtE from Thermus thermophilus (MgtE TT ) has been extensively characterized, mostly in detergent micelles, and gating-related structural dynamics in biologically relevant membranes are scarce. The MgtE homolog from Bacillus firmus (MgtE BF ) is unique since it lacks the entire Mg 2+ -sensing N-domain but has conserved structural motifs in the TM-domain for Mg 2+ transport. In this work, we have successfully purified this novel homolog in a stable and functional form, and ColabFold structure prediction analysis suggests a homodimer. Further, microscale thermophoresis experiments show that MgtE BF binds Mg 2+ and ATP, similar to MgtE TT . Importantly, we show that, despite lacking the N-domain, MgtE BF mediates Mg 2+ transport function in the presence of an inwardly directed Mg 2+ gradient in reconstituted proteoliposomes. Furthermore, comparison of the organization and dynamics of Trp residues in the TM-domain of MgtE BF in membrane mimetics, in apo- and Mg 2+ -bound forms, suggests that the cytoplasmic binding of Mg 2+ might involve modest gating-related conformational changes at the TM-domain. Overall, our results show that the gating-related structural dynamics (hydration dynamics, conformational heterogeneity) of the full-length MgtE BF is significantly changed in functionally pertinent membrane environment, emphasizing the importance of lipid-protein interactions in MgtE gating mechanisms., Competing Interests: Declaration of interests The authors declare that they do not have any conflict of interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication., Competing Interests: Declaration of Interests P.D.Z., S.M.J., and H.Z. have submitted a patent application regarding novel uses of TtAgo (PCT/US2016/025724)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Eukaryotic Argonaute proteins strictly mediate RNA-guided RNA interference. In contrast, prokaryotic Argonautes can utilize DNA guides to target complementary DNA sequences to protect their hosts against invading DNA. In this issue of Cell, Jolly and colleagues demonstrate that Thermus thermophilus Argonaute additionally participates in DNA replication by unlinking catenated chromosomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The membrane protein SecDF, belonging to the RND superfamily, enhances protein translocation at the extracytoplasmic side using a proton gradient. Here, we report the crystal structure of SecDF in a form we named Super-membrane-facing (Super F) form, demonstrating a β-barrel architecture instead of the previously reported β-sheet structure. Through this structural insight and supporting results of an in vivo crosslinking experiment, we propose a remote coupling model in which a structural change of the transmembrane region drives a functional, extracytoplasmic conformational transition., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Termination of protein synthesis on the ribosome is catalyzed by release factors (RFs), which share a conserved glycine-glycine-glutamine (GGQ) motif. The glutamine residue is methylated in vivo, but a mechanistic understanding of its contribution to hydrolysis is lacking. Here, we show that the modification, apart from increasing the overall rate of termination on all dipeptides, substantially increases the rate of peptide release on a subset of amino acids. In the presence of unmethylated RFs, we measure rates of hydrolysis that are exceptionally slow on proline and glycine residues and approximately two orders of magnitude faster in the presence of the methylated factors. Structures of 70S ribosomes bound to methylated RF1 and RF2 reveal that the glutamine side-chain methylation packs against 23S rRNA nucleotide 2451, stabilizing the GGQ motif and placing the side-chain amide of the glutamine toward tRNA. These data provide a framework for understanding how release factor modifications impact termination., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The universally conserved GTPase elongation factor G (EF-G) catalyzes the translocation of tRNA and mRNA on the ribosome after peptide bond formation. Despite numerous studies suggesting that EF-G undergoes extensive conformational rearrangements during translocation, high-resolution structures exist for essentially only one conformation of EF-G in complex with the ribosome. Here, we report four atomic-resolution crystal structures of EF-G bound to the ribosome programmed in the pre- and posttranslocational states and to the ribosome trapped by the antibiotic dityromycin. We observe a previously unseen conformation of EF-G in the pretranslocation complex, which is independently captured by dityromycin on the ribosome. Our structures provide insights into the conformational space that EF-G samples on the ribosome and reveal that tRNA translocation on the ribosome is facilitated by a structural transition of EF-G from a compact to an elongated conformation, which can be prevented by the antibiotic dityromycin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Three papers in this issue of Molecular Cell report on the structure and functional activity of type III CRISPR-Cas effector complexes, revealing novel and conserved features of the ribonucleoprotein particles that underlie prokaryotic genome defense. The new structures suggest that type I and type III complexes follow the same architectural principles and are most likely descendants of a common ancestor, the differences in RNA and protein sequences and structure of individual components notwithstanding., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Respiratory complex I, NADH:ubiquinone oxidoreductase, is a large and complex integral membrane enzyme found in respiring bacteria and mitochondria. It is responsible in part for generating the proton gradient necessary for ATP production. Complex I serves as both a proton pump and an entry point for electrons into the respiratory chain. Although complex I is one of the most important of the respiratory complexes, it is also one of the least understood, with detailed structural information only recently available. In this study, full-finite-difference Poisson-Boltzmann calculations of the protonation state of respiratory complex I in various redox states are presented. Since complex I couples the oxidation and reduction of the NADH/ubiquinone redox couple to proton translocation, the interaction of the protonation and redox states of the enzyme are of the utmost significance. Various aspects of complex I function are presented, including the redox-Bohr effect, intercofactor interactions, and the effects of both the protein dielectric and inclusion of the membrane., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

The chaperonin GroEL interacts with various proteins, leading them to adopt their correct conformations with the aid of GroES and ATP. The actual mechanism is still being debated. In this study, by use of cryo-electron microscopy, we determined the solution structure of the Thermus thermophilus GroEL-GroES complex encapsulating its substrate proteins. We observed the averaged density of substrate proteins in the center of the GroEL-GroES cavity. The position of the averaged substrate density in the cavity suggested a repulsive interaction between a majority of the substrate proteins and the interior wall of the cavity, which is suitable for substrate release. In addition, we observed a distortion of the cis-GroEL ring, especially at the position near the substrate, which indicated that the interaction between the encapsulated proteins and the GroEL ring results in an adjustment in the cavity's shape to accommodate the substrate.

Molecular dynamics simulations were carried out on Thermus thermophilus 70S ribosome with and without a nascent polypeptide inside the exit tunnel. Modeling of the polypeptide in the tunnel revealed two possible paths: one over Arg92 of L22 and one under (from the viewpoint of 50S on top of 30S). A strong interaction between L4 and Arg92 was observed without the polypeptide and when it passed over Arg92. However, when the polypeptide passed under, Arg92 repositioned to interact with Ade2059 of 23S rRNA. Using steered molecular dynamics the polypeptide could be pulled through the L4-L22 constriction when situated under Arg92, but did not move when over. These results suggest that the tunnel is closed by the Arg92-L4 interaction before elongation of the polypeptide and the tunnel leads the entering polypeptide from the peptidyl transferase center to the passage under Arg92, causing Arg92 to switch to an open position. It is possible, therefore, that Arg92 plays the role of a gate, opening and closing the tunnel at L4-L22. There is some disagreement over whether the tunnel is dynamic or rigid. At least within the timescale of our simulations conformational analysis showed that global motions mainly involve relative movement of the 50S and 30S subunits and seem not to affect the conformation of the tunnel.

Macromolecular structure determination by cryo-electron microscopy (EM) and single-particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. We describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set of its projections. We introduce a highly efficient reconstruction algorithm that is based on direct Fourier inversion and that incorporates correction for the transfer function of the microscope, thus extending the resolution limits of variance estimation. We also describe a validation method to determine the number of resampled volumes required to achieve stable estimate of the variance. The proposed bootstrap method was applied to a data set of 70S ribosome complexed with tRNA and the elongation factor G. The proposed method of variance estimation opens new possibilities for single-particle analysis, by extending applicability of the technique to heterogeneous data sets of macromolecules and to complexes with significant conformational variability.

The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba3 enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A3 are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A3. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.

Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.

Asparagine, one of the 22 genetically encoded amino acids, can be synthesized by a tRNA-dependent mechanism. So far, this type of pathway was believed to proceed via two independent steps. A nondiscriminating aspartyl-tRNA synthetase (ND-DRS) first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase (AdT), which then converts the tRNA-bound aspartate into asparagine. We show herein that the ND-DRS, tRNAAsn, and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process. Our results indicate that the tRNAAsn-mediated linkage between the ND-DRS and AdT enables channeling of the mischarged aspartyl-tRNAAsn intermediate between DRS and AdT active sites to prevent challenging of the genetic code integrity. We propose that formation of a ribonucleoprotein is a general feature for tRNA-dependent amino acid biosynthetic pathways that are remnants of earlier stages when amino acid synthesis and tRNA aminoacylation were coupled.

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.

ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.

The AAA(+) chaperone ClpB mediates the reactivation of aggregated proteins in cooperation with the DnaK chaperone system. ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel. Its unique middle (M) domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization. Here, we demonstrate that the conserved helix 3 of the M domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel. Helix 3 exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states. This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity.

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.

Two mesophilic/thermophilic variants of the G-domain of the elongation factor Tu were studied via molecular dynamics simulations. By analyzing the simulation data via the Voronoi space tessellation, we have found that the two proteins have the same macromolecular packing, while the water-exposed surface area is larger for the thermophile. A larger coordination with water is probably due to a peculiar corrugation of the exposed surface of this species. From an enthalpic point of view, the thermophile shows a larger number of intramolecular hydrogen bonds, stronger electrostatic interactions, and a flatter free-energy landscape. Overall, the data suggest that the specific hydration state enhances macromolecular fluctuations but, at the same time, increases thermal stability.

A general method is presented that allows the separation of the rigid body motions from the nonrigid body motions of structural subunits when bound in a complex. The application presented considers the motions of the tRNAs: free, bound to the ribosome and to a synthase. We observe that both the rigid body and nonrigid body motions of the structural subunits are highly controlled by the large ribosomal assembly and are important for the functional motions of the assembly. For the intact ribosome, its major parts, the 30S and the 50S subunits, are found to have counterrotational motions in the first few slowest modes, which are consistent with the experimentally observed ratchet motion. The tRNAs are found to have on average approximately 72-75% rigid body motions and principally translational motions within the first 100 slow modes of the complex. Although the three tRNAs exhibit different apparent total motions, after the rigid body motions are removed, the remaining internal motions of all three tRNAs are essentially the same. The direction of the translational motions of the tRNAs are in the same direction as the requisite translocation step, especially in the first slowest mode. Surprisingly the small intrinsically flexible mRNA has all of its internal motions completely inhibited and shows mainly a rigid-body translation in the slow modes of the ribosome complex. On the other hand, the required nonrigid body motions of the tRNA during translocation reveal that the anticodon-stem-loop, as well as the acceptor arm, of the tRNA enjoy a large mobility but act as rigid structural units. In summary, the ribosome exerts its control by enforcing rigidity in the functional parts of the tRNAs as well as in the mRNA.

Ribosome recycling factor is proposed to be flexible, and that flexibility is believed to be important to its function. Here we use molecular dynamics to test the flexibility of Escherichia coli RRF (ecRRF) with and without decanoic acid bound to a hydrophobic pocket between domains 1 and 2, and Thermus thermophilus RRF (ttRRF) with and without a mutation in the hinge between domains 1 and 2. Our simulations show that the structure of ecRRF rapidly goes from having an interdomain angle of 124 degrees to an angle of 98 degrees independently of the presence of decanoic acid. The simulations also show that the presence or absence of decanoic acid leads to changes in ecRRF flexibility. Simulations of wild-type and mutant ttRRF (R32G) show that mutating Arg-32 to glycine decreases RRF flexibility. This was unexpected because the range of dihedral angles for arginine is limited relative to glycine. Furthermore, the interdomain angle of wild-type T. thermophilus goes from 81 degrees to 118 degrees whereas the R32G mutant remains very close to the crystallographic angle of 78 degrees . We propose that this difference accounts for the fact that mutant ttRRF complements an RRF deficient strain of E. coli whereas wild-type ttRRF does not. When the ensemble of RRF structures is modeled into the ribosomal crystal structure, a series of overlaps is found that corresponds with regions where conformational changes have been found in the cryoelectron microscopic structure of the RRF/ribosome complex, and in the crystal structure of a cocomplex of RRF with the 50S subunit. There are also overlaps with the P-site, suggesting that RRF flexibility plays a role in removing the deacylated P-site tRNA during termination of translation.

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.

Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, we show that ribosomes are able to disrupt downstream helices, including a perfect 27 base pair helix of predicted T(m) = 70 degrees . Using helices of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. We conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.

The tRNA(Gm18) methyltransferase (TrmH) catalyzes the 2'-O methylation of guanosine 18 (Gua18) of tRNA. We solved the crystal structure of Thermus thermophilus TrmH complexed with S-adenosyl-L-methionine at 1.85 A resolution. The catalytic domain contains a deep trefoil knot, which mutational analyses revealed to be crucial for the formation of the catalytic site and the cofactor binding pocket. The tRNA dihydrouridine(D)-arm can be docked onto the dimeric TrmH, so that the tRNA D-stem is clamped by the N- and C-terminal helices from one subunit while the Gua18 is modified by the other subunit. Arg41 from the other subunit enters the catalytic site and forms a hydrogen bond with a bound sulfate ion, an RNA main chain phosphate analog, thus activating its nucleophilic state. Based on Gua18 modeling onto the active site, we propose that once Gua18 binds, the phosphate group activates Arg41, which then deprotonates the 2'-OH group for methylation.

The ring-forming molecular chaperone Hsp104/ClpB is a member of the AAA+ protein family which rescues proteins from aggregated states. The newly determined crystal structure of ClpB provides new insights into the mechanism of protein disaggregation, suggesting a crowbar activity mediated by a unique coiled-coil domain.

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.

Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the post-termination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.

Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.

The recently solved crystal structures of Thermus thermophilus elongation factor G, with and without GDP, reveal a protein of five domains with surprising features which can be correlated with biochemical data to suggest probable functional roles.