The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution.

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6Å resolution and provide insight into the RNA geometry and hydration.

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices.

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA(Gly)/glycyl-tRNA sythetase system is member of the so-called 'class II system' in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence. In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA(Gly) acceptor stem microhelices and their surrounding hydration patterns.

Crystal structure of a human tRNA(Gly) microhelix at 1.2A resolution.

The tRNA(Gly)/glycyl-tRNA synthetase (GlyRS) system belongs to the so-called 'class II aminoacyl-tRNA synthetase system' in which tRNA identity elements are assured by rather few and simple determinants mostly located in the tRNA acceptor stem. Regarding evolutionary aspects, the tRNA(Gly)/GlyRS system is a special case. There exist two different types of GlyRS, namely an archaebacterial/human type and a eubacterial type reflecting an evolutionary divergence within this system. Here we report the crystal structure of a human tRNA(Gly) acceptor stem microhelix at 1.2A resolution. The local geometric parameters of the microhelix and the water network surrounding the RNA are presented. The structure complements the previously published Escherichia coli tRNA(Gly) aminoacyl stem structure.

The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins.

The X-ray structure at 2.7A resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.

Crystal structure of an Escherichia coli tRNA(Gly) microhelix at 2.0 A resolution.

tRNA identity elements determine the correct aminoacylation by the cognate aminoacyl-tRNA synthetase. In class II aminoacyl tRNA synthetase systems, tRNA specificity is assured by rather few and simple recognition elements, mostly located in the acceptor stem of the tRNA. Here we present the crystal structure of an Escherichia coli tRNA(Gly) aminoacyl stem microhelix at 2.0 A resolution. The tRNA(Gly) microhelix crystallizes in the space group P3(2)21 with the cell constants a=b=35.35 A, c=130.82 A, gamma=120 degrees . The helical parameters, solvent molecules and a potential magnesium binding site are discussed.

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex.

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif.

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Nucleic acid-based modulation of cardiac gene expression for the treatment of cardiac diseases. Approaches and perspectives.

During the past few years major conceptual and technical advances have been made towards the therapeutic modulation of cardiac gene expression for the treatment of cardiac diseases. Among these are 1) the identification of new molecular therapy targets in cardiac disorders, often derived from genetic animal models. 2) A better understanding of the molecular and cellular determinants of cardiac gene transfer in vivo, in animal models and in first clinical trials. 3) The development of novel regulatable and long-term stable vector systems. This review is focused on nucleic acid-based modulation of cardiac calcium homeostasis as a paradigm for the new gene therapeutic approaches, since recent landmark papers have suggested this to be a molecular target of key importance in heart failure. In particular, the development of severe heart failure in the genetic MLP(-/-) animal model could be completely abolished by the targeted ablation of phospholamban (PL), a key regulator of cardiac calcium homeostasis. This impressive effect of permanent germline PL ablation provides-in conjunction with former important work on disturbed calcium handling in the failing human heart-a rationale for the gene therapeutic approach of ad hoc suppression of PL by antisense strategies (antisense RNAs, ribozymes, RNA interference) or PL variants. Based on the broad spectrum of methods employed to characterize this general strategy, PL-targeted approaches may be considered as a paradigm of future genetic treatments of cardiac disorders, although the differences between animal models and humans must be kept in mind. High safety of any such therapy will be a prerequisite for any possible clinical application and therefore novel methods to improve control are being devised: 1) The regulation of gene therapy vectors by biochemical abnormalities associated with the target disease itself (" Induction-by-Disease" gene therapy). 2) External control of vector activity by the employment of drug-sensitive promotors. In addition, the important goal of cardiac long-term stability of the therapeutic vectors has recently been achieved in animal models using vectors derived from adeno-associated viruses (AAVs).

First look at RNA in L-configuration.

Nucleic acid molecules in the mirror image or L-configuration are unknown in nature and are extraordinarily resistant to biological degradation. The identification of functional L-oligonucleotides called Spiegelmers offers a novel approach for drug discovery based on RNA. The sequence r(CUGGGCGG).r(CCGCCUGG) was chosen as a model system for structural analysis of helices in the L-configuration as the structure of the D-form of this sequence has previously been determined in structural studies of 5S RNA domains, in particular domain E of the Thermus flavus 5S rRNA [Perbandt et al. (2001), Acta Cryst. D57, 219-224]. Unexpectedly, the results of crystallization trials showed little similarity between the D- and the L-forms of the duplex in either the crystallization hits or the diffraction performance. The crystal structure of this L-RNA duplex has been determined at 1.9 A resolution with R(work) and R(free) of 23.8 and 28.6%, respectively. The crystals belong to space group R32, with unit-cell parameters a = 45.7, c = 264.6 A. Although there are two molecules in the asymmetric unit rather than one, the structure of the L-form arranges helical pairs in a head-to-tail fashion to form pseudo-continuous infinite helices in the crystal as in the D-form. On the other hand, the wobble-like G.C(+) base pair seen in the D-RNA analogue does not appear in the L-RNA duplex, which forms a regular double-helical structure with typical Watson-Crick base pairing.

Crystallization and structure analysis of Thermus flavus 5S rRNA helix B.

The crystallization conditions of the synthetic RNA duplex r(GCGGCGU)*r(GCGCCGC), part of the Thermus flavus 5S rRNA domain B, were investigated in detail. The crystallization analysis revealed a relative narrow crystallization zone. Single sequence variations did not enhance the crystal quality, however the crystallization under microgravity provided crystals of higher quality. They belong to the space group P3(1)21 with unit cell dimensions of a = b = 35.0 A and c = 141.2 A. Diffraction data up to 2.6 A were collected and the structure subsequently analysed and refined to an R-value of 22.4 %. The conformation of the two molecules in the asymmetric unit is stabilized by intermolecular hydrogen bonds. The two molecules A and B are perpendicular to each other and interacting head to tail with symmetry related molecules. They form pseudo-continuous infinite helices in the crystal lattice.

The cell-free protein biosynthesis--applications and analysis of the system.

The in vitro protein biosynthesis has the potentials to become a powerful technology for biochemical research. Beside the determination of structure and function the in vitro evolution of proteins is also of great interest. The system described was used to produce bovine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag II affinity peptide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selection of a specific mRNA coding for a shortened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. After nine cycles of transcription, translation, affinity selection and RT-PCR the protein with the His-tag could be enriched 10(8)-fold. In order to correlate a possible relationship between changes in protein population and biological function studies were initiated in which 2-dimensional protein patterns of the total in vitro system were compared after 0 and 2 h reaction time. The very interesting findings are that a number of proteins disappear, while others are newly formed during protein synthesis.

Regulatory RNAs.

In addition to mRNA, rRNA and tRNA, which play central roles within cells, there are a number of regulatory, non-coding RNAs (ncRNAs). Of varying lengths, ncRNAs have no long open reading frame. While not encoding proteins, they may act as riboregulators, and their main function is posttranscriptional regulation of gene expression. Many ncRNAs have been identified and characterized both in prokaryotes and eukaryotes, and are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing, controlling cell growth and differentiation. Some are associated with the abnormalities in imprinted inheritance that occur in several well-known developmental and neurobehavioral disorders. Other ncRNAs accomplish regulation by modulating the activity of proteins. Several rRNAs are able to sustain enzymatic reactions implicated in the translation process including synthesis of peptide bonds within the ribosome. The different roles played by widely distributed RNAs acting in diverse ways, suggest the flexibility and versality of these molecules in regulatory processes. This review summarizes the available biochemical and structural data on known regulatory RNAs.

In vitro selection of other proteins than antibodies by means of ribosome display.

The in vitro selection and simultaneous evolution of proteins is feasible by means of ribosome display. Here, we describe the use of a protein bearing a binding property without being an antibody for affinity enrichment of the ternary complex, consisting of a protein, a ribosome and a encoding mRNA. The binding property was a simple affinity tag, namely Strep-tag II and His-tag. We could demonstrate that the selection of a specific mRNA encoding a shortened bovine heart fatty acid binding protein with a N-terminal His-tag was possible. After nine cycles of transcription, translation, affinity selection and reverse transcription PCR the protein with the His-tag could be enriched 10(8)-fold.

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.

The specific hydrolysis of HIV-1 TAR RNA element with the anti-TAR hammerhead ribozyme: structural and functional implications.

The main transcriptional regulator of the human immunodeficiency virus is the Tat protein, which recognises and binds to a fragment RNA at the 5' end of viral mRNA, named transactivation response element (TAR) RNA. Extensive mutagenesis studies have shown that a region of TAR RNA important for Tat binding involves a set of nucleotides surrounding a characteristic UCU nucleotide bulge. The specific Tat-TAR complex formation enhances the rate of transcription elongation but inhibition of that interaction prevents the human immunodeficiency virus type 1 (HIV-1) replication. If so, a possibility of virus inactivation would be a site specific degradation of the TAR RNA element. To break down and inactivate TAR RNA, we designated the anti-hammerhead (HH) ribozyme to cleave nucleosides within the bulge. We showed for the first time the new type of the AUC hammerhead ribozyme, which hydrolyses specifically the TAR RNA element at C8 nucleotide in the bulge (C24 in the standard TAR RNA numbering). The cleavage reaction has broad magnesium requirements. Mn and particularly Ca are less efficient. Argininamide interferes with the cleavage of TAR RNA induced by the ribozyme. These results have two implications; (i) structural, where the HIV-1 TAR RNA element in solution occurs in equilibrium of only two forms, one of which, a double stranded RNA, meets structural requirements for ribozyme pairing and cleavage, and (ii) functional, the HH ribozyme can be explored for an inactivation of HIV-1 through the TAR RNA element deintegration.

Structure of an RNA duplex with an unusual G.C pair in wobble-like conformation at 1.6 A resolution.

The structure of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) has been determined at 1.6 A resolution and refined to a final R factor of 18.3% (R(free) = 24.1%). The sequence of the RNA fragment resembles domain E of Thermus flavus 5S rRNA. A previously undescribed wobble-like G.C base-pair formation is found. Owing to the observed hydrogen-bond network, it is proposed that the cytosine is protonated at position N3. The unusual base-pair formation is presumably strained by intermolecular interactions. In this context, crystal packing and particular intermolecular contacts may have direct influence on the three-dimensional structure. Furthermore, this structure includes two G.U wobble base pairs in tandem conformation, with the purines forming a so-called 'cross-strand G stack'.

The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage.

The structure of native yeast tRNA(Phe) and wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg+2 can induce significant changes in the hydroxyl radical cleavage pattern of tRNA(Phe). It also turned out that a reactivity of tRNAz(Phe) towards *OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg2+ binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNA(Phe) although some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg+2 binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with *OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.

An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs.

The structure-function relationship in RNA molecules is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an analysis of the distribution of G-U pairs in eukaryotic 5S ribosomal RNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.