Tertiary Core Research Articles

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

Riboswitches are highly conserved RNA elements that located in the 5'-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

Thermophilic Enzymes

Substantial improvements in the industrial production of goods led to a widespread feeling of unlimited access to food, commodities, and energy. As greener alternatives for industrial processes are in demand, scientists have turned to enzymes, looking for apt biocatalysts. Focusing on extremophiles, this mini review draws a comparison between thermophiles and their mesophilic counterparts, exploring what features are instrumental to their thermostability. A higher number of ion-pairs, hydrophobicity of buried side chains, compact tertiary structure cores, and a complex network of hydrogen bonds are the four main characteristics responsible for the robustness of thermophilic enzymes.

Experimental Investigation of Polymer-Coated Silica Nanoparticles for Enhanced Oil Recovery.

Recently, polymer-coated nanoparticles were proposed for enhanced oil recovery (EOR) due to their improved properties such as solubility, stability, stabilization of emulsions and low particle retention on the rock surface. This work investigated the potential of various polymer-coated silica nanoparticles (PSiNPs) as additives to the injection seawater for oil recovery. Secondary and tertiary core flooding experiments were carried out with neutral-wet Berea sandstone at ambient conditions. Oil recovery parameters of nanoparticles such as interfacial tension (IFT) reduction, wettability alteration and log-jamming effect were investigated. Crude oil from the North Sea field was used. The concentrated solutions of PSiNPs were diluted to 0.1 wt % in synthetic seawater. Experimental results show that PSiNPs can improve water flood oil recovery efficiency. Secondary recoveries of nanofluid ranged from 60% to 72% of original oil in place (OOIP) compared to 56% OOIP achieved by reference water flood. In tertiary recovery mode, the incremental oil recovery varied from 2.6% to 5.2% OOIP. The IFT between oil and water was reduced in the presence of PSiNPs from 10.6 to 2.5–6.8 mN/m, which had minor effect on EOR. Permeability measurements indicated negligible particle retention within the core, consistent with the low differential pressure observed throughout nanofluid flooding. Amott–Harvey tests indicated wettability alteration from neutral- to water-wet condition. The overall findings suggest that PSiNPs have more potential as secondary EOR agents than tertiary agents, and the main recovery mechanism was found to be wettability alteration.

Probing selected structural regions in the secreted phospholipase A2 from Arabidopsis thaliana for their impact on stability and activity

In contrast to the well characterized secreted phospholipases A2 (sPLA2) from animals, their homologues from plants have been less explored. Their production in purified form is more difficult, and no data on their stability are known. In the present paper, different variants of the sPLA2 isoform α from Arabidopsis thaliana (AtPLA2α) were designed using a new homology model with the aim to probe the impact of regions that are assumed to be important for stability and catalysis. Moreover tryptophan residues were introduced in critical regions to enable stability studies by fluorescence spectroscopy. The variants were expressed in Escherichia coli and the purified enzymes were analyzed to get first insights into the peculiarities of structure stability and structure activity relationships in plant sPLA2s in comparison with the well-characterized homologous enzymes from bee venom and porcine pancreas. Stability data of the AtPLA2 variants obtained by fluorescence or CD measurements of the reversible unfolding by guanidine hydrochloride and urea showed that all enzyme variants are less stable than the enzymes from animal sources although a similar tertiary core structure can be assumed based on molecular modeling. More extended loop structures at the N-terminus in AtPLA2α are suggested to be the main reasons for the much lower thermodynamic stabilities and cooperativities of the transition curves. Modifications in the N-terminal region (insertion, deletion, substitution by a Trp residue) exhibited a strong positive effect on activity whereas amino acid exchanges in other regions of the protein such as the Ca(2+)-binding loop and the loop connecting the two central helices were deleterious with respect to activity.

TRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA(Ala)GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA(Ala)GGC sequences. This suggests that the sequences of bacterial tRNA(Ala)GGC have evolved to avoid reading incorrect codons.

The Test of Amine Polymeric Alcohol Drilling Fluid System in Baka Block

2500 meters below Baka block appears extremely thick coal seam and distributed long, unconsolidated and mixed up with carbonaceous mudstone between beds. The relieved stress after a new borehole is drilled will lead to the wellbore caving and peeling and finally form an approximate elliptic borehole, causing vicious accidents such as bit bouncing, bit freezing while tripping and even borehole discarding. The anti-sloughing capability of Amine Polymeric Alcohol (AP-1) was evaluated by tertiary recovery test and core immersing test, the formula of Amine Polymeric Alcohol drilling fluid system was also optimized. Indoor research shows that the drilling fluid is of strong inhibition and pollution resistance. Its plastic viscosity is 24 mPa•s, shear force is 11 Pa and permeability recovery value reached（81.9-89.6）%. The successful test in Ke 21 Ping 1 well indicates that Amine Polymeric Alcohol drilling fluid system with powerful inhibition can effectively solve the collapse of Jurassic coal seam and carbonaceous mudstone, present high lubricity and can improve the drilling speed effectively, meanwhile, it performances well in gaining reservoir protection, controlling drilling fluid rheological properties, enhancing pollution resistance and reducing the occurrence of complex accidents downhole. Thus it can provide reference for future drilling operation in sloughing formations.

Stroke Associated Infection: A Prospective Hospital-Based Study at Tertiary Care Institution of North India

BACKGROUND: Immune dysfunction is increasingly recognized to be contributing to stroke associated infections.
 OBJECTIVE: The present study was aimed to study the magnitude of the stroke associated infection in a mixed population of hemorrhagic and ischemic strokes and its predisposing f actors.
 MATERIAL &METHODS: The study population consisted of 173 consecutive stroke admissions at SKIMS Srinagar, a tertiary core hospital. The patients developing fever at admission or in hospital were included and underwent neuroimaging and workup for the source of infection. The follow up was done upto death or discharg e of the patient. A univariate and binary logistic regression analysis was done to find the patient characteristics associated with infection.
 RESULTS: The stroke subtypes in the study were 60.1% hemorrhagic and 39.9% ischemic strokes. The majority of hemorrhagic strokes (65A%) were located in putamina while as anterior circulation territory contributed about 80% of the ischemic strokes. Fifty eight (33.5%) patients developed stroke associated infection. Chest was the source of infection in 58.6% of the cases. The culture positivity rate was poor (18.9%) in the patient study. The presence of a comorbid illness {OR 3.4), volume of hematoma (OR 4,7). and ventricular extension of the hemorrhoge (OR 4.9) significantly increased the odds ratio of stroke associated infection.
 CONCLUSIONS: The frequency of post stroke infection is around 33%, chest being the commonest source. Presence of a comorbid illness, volume of hemorrhage a nd ventricular extension increase the risk of infection . JMS 2012; 15(1):22-27

Potential for interdependent development of tRNA determinants for aminoacylation and ribosome decoding

Although the nucleotides in tRNA required for aminoacylation are conserved in evolution, bacterial aminoacyl-tRNA synthetases (aaRSs) are unable to acylate eukaryotic tRNA. The cross-species barrier may be due to the absence of eukaryote-specific domains from bacterial aaRSs. Here we show that while E. coli CysRS cannot acylate human tRNACys, the fusion of a eukaryote-specific domain of human CysRS overcomes the cross-species barrier in human tRNACys. In addition to enabling recognition of the sequence differences in the tertiary core of tRNACys, the fused eukaryotic domain redirects the specificity of E. coli CysRS from the A37 present in bacterial tRNACys to the G37 in mammals. Further experiments show that the accuracy of codon recognition on the ribosome was also highly sensitive to the A37-to-G37 transition in tRNACys. These results raise the possibility of the development of tRNA nucleotide determinants for aminoacylation being interdependent with those for ribosome decoding.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Heat Maps for Intramolecular Communication in an RNP Enzyme Encoding Glutamine

Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.

Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition.

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNAGln and Asn-tRNAAsn, proceeds by an indirect route in which mischarged Glu-tRNAGln or Asp-tRNAAsn is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNAGln docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNAAsn from Asp-tRNAAsp in archaea. Furthermore, we identified a 310 turn in GatB that permits the bacterial GatCAB to distinguish a U1–A72 base pair from a G1–C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1–C72 base pairs.

Crystal structure of human selenocysteine tRNA

Selenocysteine (Sec) is the 21st amino acid in translation. Sec tRNA (tRNASec) has an anticodon complementary to the UGA codon. We solved the crystal structure of human tRNASec. tRNASec has a 9-bp acceptor stem and a 4-bp T stem, in contrast with the 7-bp acceptor stem and the 5-bp T stem in the canonical tRNAs. The acceptor stem is kinked between the U6:U67 and G7:C66 base pairs, leading to a bent acceptor-T stem helix. tRNASec has a 6-bp D stem and a 4-nt D loop. The long D stem includes unique A14:U21 and G15:C20a pairs. The D-loop:T-loop interactions include the base pairs G18:U55 and U16:U59, and a unique base triple, U20:G19:C56. The extra arm comprises of a 6-bp stem and a 4-nt loop. Remarkably, the D stem and the extra arm do not form tertiary interactions in tRNASec. Instead, tRNASec has an open cavity, in place of the tertiary core of a canonical tRNA. The linker residues, A8 and U9, connecting the acceptor and D stems, are not involved in tertiary base pairing. Instead, U9 is stacked on the first base pair of the extra arm. These features might allow tRNASec to be the target of the Sec synthesis/incorporation machineries.

A cytidine deaminase edits C to U in transfer RNAs in Archaea.

All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA's tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the "cytidine deaminase-like" superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs.

Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

All mature tRNA molecules have the conserved CCA sequence at the 3' end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 3' end. Using Escherichia coli tRNA(Cys) as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA(Cys), separated from the unlabeled precursor tRNA by reverse phase high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNA(Cys) with E. coli CysRS reveals an equilibrium K(d) closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyC-labeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

Complementary Structural Mass Spectrometry Techniques Reveal Local Dynamics in Functionally Important Regions of a Metastable Serpin

Serpins display a number of highly unusual structural properties along with a unique mechanism of inhibition. Although structures of numerous serpins have been solved by X-ray crystallography, little is known about the dynamics of serpins in their inhibitory active conformation. In this study, two complementary structural mass spectrometry methods, hydroxyl radical-mediated footprinting and hydrogen/deuterium (H/D) exchange, were employed to highlight differences between the static crystal structure and the dynamic conformation of human serpin protein, alpha(1)-antitrypsin (alpha(1)AT). H/D exchange revealed the distribution of flexible and rigid regions of alpha(1)AT, whereas footprinting revealed the dynamic environments of several side chains previously identified as important for the metastability of alpha(1)AT. This work provides insights into the unique structural design of alpha(1)AT and improves our understanding of its unusual inhibition mechanism. Also, we demonstrate that the combination of the two MS techniques provides a more complete picture of protein structure than either technique alone.

Indirect Readout of tRNA for Aminoacylation

Aminoacylation of tRNA by aminoacyl-tRNA synthetases is the essential reaction that matches protein amino acids with the trinucleotide sequences specified in mRNA. Direct electrostatic interactions made by tRNA synthetases with discriminating functional groups on the tRNA bases have long been known to determine aminoacylation specificity. However, structural and biochemical studies have revealed a second "indirect readout" mechanism that makes an important contribution as well. In indirect readout, the sequence-dependent conformations of tRNA are recognized through protein contacts with the sugar-phosphate backbone and with nonspecific portions of the bases. This mechanism appears to function in single-stranded regions, in canonical A-type duplex segments, and in the complex tertiary core portion of the tRNA. Operation of the indirect mechanism is not exclusive of the direct mechanism, and both are further mediated by induced-fit rearrangements, in which enzyme and tRNA undergo precise conformational changes after formation of an initial encounter complex. The examples of indirect readout in tRNA synthetase complexes extend the concept beyond its traditional application to DNA duplexes and serve as models for the operation of this mechanism in more complex systems such as the ribosome.

Mechanism of the Difference in the Binding Affinity of E. coli tRNA Gln to Glutaminyl-tRNA Synthetase Caused by Noninterface Nucleotides in Variable Loop

Aminoacyl-tRNA synthetases (ARSs) distinguish their cognate tRNAs from many other kinds of tRNAs, despite the very similar tertiary structures of tRNAs. Many researchers have supported the view that this recognition is achieved by intermolecular interactions between tRNA and ARS. However, one of the aptamers of Escherichia coli glutamine specific tRNA, var-AGGU, has a higher affinity to ARS than the wild-type, although the sequence difference only lies in the variable loop located on the opposite side of the binding interface with ARS. To understand the reason for the difference in affinity, we did molecular dynamics simulations on tRNAs and their complexes with ARS. We calculated the enthalpic and entropic contributions to the binding free energy with the molecular mechanics-Poisson-Boltzmann/surface area method and found that the entropic difference plays an important role in the difference in binding free energies. During the molecular dynamics simulations, dynamic rearrangements of hydrogen bonds occurred in the tertiary core region of the wild-type tRNA, whereas they were not observed in the free var-AGGU simulation. Since the internal mobility was suppressed upon complex formation with ARS, the entropy loss in the wild-type was larger than that of the aptamer. We therefore concluded that the sequence difference in the variable loop caused the difference in the internal mobility of the tertiary core region tRNAs and led to the difference in the affinity to ARS through the entropy term.