T-psi-C: user friendly database of tRNA sequences and structures.

tRNAs have been widely studied for their role as genetic code decoders in the ribosome during translation, but have recently received new attention due to the discovery of novel roles beyond decoding, often in connection with human diseases. Yet, existing tRNA databases have not been updated for more than a decade, so they do not contain this new functional information and have not kept pace with the rate of discovery in this field. Therefore, a regularly updated database that contains information about newly discovered characteristics of tRNA molecules and can be regularly updated is strongly needed. Here, we report the creation of the T-psi-C database (http://tpsic.igcz.poznan.pl), an up-to-date collection of tRNA sequences that contains data obtained from high-throughput tRNA sequencing, e.g. all isoacceptors and isodecoders for human HEK293 cells. This database also contains 3D tRNA structures obtained from Protein Data Bank and generated using homology modeling. The T-psi-C database can be continuously updated by any member of the scientific community, and contains its own application programming interface (API), which allows users to retrieve or upload data in JSON format. Altogether, T-psi-C is user-friendly, easy to develop and an up-to-date source of knowledge about tRNAs.

Positive and negative regulation of vertebrate separase by Cdk1-cyclin B1 may explain why securin is dispensable.

Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin.

A lateral flow assay for identification of Escherichia coli by ribosomal RNA hybridisation.

Existing technologies for analysis of microbiological contaminants in food or clinical samples are often expensive and require laboratory settings and trained personnel. Here we present a lateral flow assay employing gold nanoparticle-oligodeoxynucleotide conjugates and four-component sandwich hybridisation for direct detection of specific sequences in bacterial 16S ribosomal RNA. Combined with rapid "one step" lysis the developed procedure allows detection of 5 × 10(4) colony forming units per mL Escherichia coli within less than 25 minutes. Several Escherichia coli strains were detected successfully, whereas non-related as well as closely related bacterial species produced no signal. The developed nucleic acid lateral flow assay is inexpensive, rapid to perform and requires no nucleic acid amplification step.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.

Electrochemical detection of microRNAs via gap hybridization assay.

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Detection of Escherichia coli in meat with an electrochemical biochip.

Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli, were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h.

Simultaneous and site-directed incorporation of an ester linkage and an azide group into a polypeptide by in vitro translation.

A method is presented by which an azide-containing side chain can be introduced into any internal position of a polypeptide chain by in vitro translation. For this, 2'-deoxy-cytidylyl-(3'-->5')-adenosine was acylated on the 3'(2')-hydroxyl group of adenosine with 6-azido-2(S)-hydroxyhexanoic acid (AHHA), an alpha-hydroxy- and epsilon-azide derivative of L-lysine. The acylated dinucleotide was enzymatically ligated with a tRNA transcript to provide chemically stable E. coli suppressor AHHA-tRNA(Cys(CUA)). The esterase 2 gene from Alicyclobacillus acidocaldarius was modified by the amber stop codon (UAG) on position 118. Using AHHA-tRNA(Cys(CUA)) in an E. coli in vitro translation/transcription system, the site-directed introduction of an azide group linked to a backbone ester into the esterase polypeptide was achieved. The yield of the synthesized modified protein reached 80% compared to translation of the native esterase. Subsequently, azide coupling with an alkyne-modified oligodeoxynucleotide demonstrated the feasibility of this approach for conjugation of polypeptides.

Detection of bacterial 16S rRNA using multivalent dendrimer-reporter enzyme conjugates.

Novel enzyme-oligodeoxynucleotide conjugate was synthesized to improve sensitivity of Escherichia coli 16S rRNA detection on gold electrodes. Thermostable esterase 2 from Alicyclobacillus acidocaldarius was multiply conjugated to a polyamidoamine dendrimer functionalized by one universal detector oligodeoxynucleotide. Three components rRNA/DNA hybridization between capture oligodeoxynucleotide covalently immobilized on a gold electrode, 16S rRNA and the multivalent esterase-dendrimer cluster was used for detection of E. coli. The linear dependence of the electrochemical signals to analyte concentration revealed a detection limit of 50 colony forming units E. coli, which represents a tenfold signal enhancement if compared to the detection limit achieved with monovalent esterase-oligodeoxynucleotide conjugate.

Correction of errors during protein synthesis.

Quality control: The incorporation of a wrong amino acid into a growing polypeptide chain induces a correction step in which the release factor (RF1) hydrolyzes the peptide from the incorrectly matched peptidyl-tRNA (see picture). The nascent erroneous polypeptide is released from the ribosome and degraded.

Rapid, specific and sensitive electrochemical detection of foodborne bacteria.

Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.

tRNAdb 2009: compilation of tRNA sequences and tRNA genes.

One of the first specialized collections of nucleic acid sequences in life sciences was the 'compilation of tRNA sequences and sequences of tRNA genes' (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs.

Enhancement of electrochemical signal on gold electrodes by polyvalent esterase-dendrimer clusters.

5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.

Multidomain initiation factor 2 from Thermus thermophilus consists of the individual autonomous domains.

The three-dimensional chalice-like crystal structure of initiation factor 2 IF2/eIF5B from Methanobacterium thermoautotrophicum represents a novel fold and domain architecture in which the N-terminal G domain and the C-terminal C domain are separated by an approximately 40 A alpha-helix. Homologous Thermus thermophilus initiation factor 2 (IF2wt), G (IF2G), and C (IF2C) domains were successfully overexpressed and purified which enabled us to perform a thermodynamic analysis and to asses the role of the domain architecture in this atypical fold. Circular dichroism in the far-UV region demonstrated that the proteins are well-folded and that the secondary structure content resembles that of IF2 from M. thermoautotrophicum. IF2wt and IF2G are monomeric proteins, while IF2C has a tendency to form dimeric species as shown by sedimentation velocity studies on analytical ultracentrifugation and differential scanning calorimetry scan analysis. Thermal denaturation studies of multidomain IF2wt reveals an exceptionally high reversibility (>90%) of the transition with a melting temperature of 94.5 degrees C. Melting temperature of IF2wt may be further increased in the presence of its physiological ligand GDP and the GTP analogue, GppNHp. The high reversibility of denaturation is achieved by the modular structure of the protein and by the high reversibility of the thermal denaturation of IF2G. On the other hand, hydrophobic IF2C aggregates during the thermal transition, and the aggregation is suppressed by guanidine hydrochloride. Isothermal denaturation demonstrates that both IF2G and IF2C have comparable stabilities of 46 and 33 kJ/mol, respectively. The apparent cooperative unfolding of the full-length protein has an unusually small denaturant m value. This together with the phase diagram method of analysis indicates the presence of intermediate(s) due to the independent unfolding of IF2G and IF2C. Despite an absence of apparent interactions between the domains in vitro, IF2G plays a role in IF2C reversibility in thermal denaturation. In conclusion, interactions between the domains of folded IF2wt in vivo are likely mediated by their alpha-helix connection and/or by a conformational change on the ribosome.

Flexibility and enzyme activity of NADH oxidase from Thermus thermophilus in the presence of monovalent cations of Hofmeister series.

Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na(+), Gdm(+), NH(4)(+), Li(+), K(+) and Cs(+)) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis-Menten constants for NADH, accessibility of the active site as reflected by Stern-Volmer constants and activity of NOX at high cation concentrations (1-2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465-476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters.

Ligand-directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy.

Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.

Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides.

A novel dual function (reporter and affinity) tag system has been developed. Expression vectors have been constructed to express polypeptides in Escherichia coli cells as C-terminal fusions with esterase 2, a 34-kDa protein from Alicyclobacillus acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotometrically or by activity staining in the polyacrylamide gels. The fusion proteins can be purified from crude bacterial extracts under non-denaturing conditions by one step affinity chromatography on Sepharose CL-6B immobilized trifluoromethyl-alkyl-ketone. The esterase carrier can be cleaved from fusion proteins by digestion with amino acid sequence-specific proteases blood coagulation factor Xa. The system has been used successfully for the expression and purification of polypeptides from different prokaryotic and eukaryotic organisms.