Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.     

5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.     

A common sugar as model for many-sided natural product modification. From D-fructose via Dtagatose to 2-C-chlorodifluoro-methylated D-arabinopyranos-5-ulose derivatives

Starting with 3,4-O- [(R)-2,2,2-trichloroethylidene]-1,2-O-isopropylidene- beta-D- tagatopyranose 2 obtained from 1,2-O-isopropylidene-beta-D-fructopyranose 1 by a non-classical one-step acetalization with chloral/DC~, the fluoroalkylated glyeosyl donors 15 and 17 were synthesised in 3~4 steps. By this sequence, one stereogenic center was inverted, one new chiral center was introduced, and one stereogenic center, for the time being eliminated, was later re-introduced. The glycals 11 and 12, key intermediates of the synthesis sequence, were accessible from triflate precursors (e. g., 10) by treatment with DBU. Corresponding halogeno-(6,7), tosyl-(5, 8), or mesyl-(9) precursors were unsuitable. The stereaselective introduction of a chlorodifluoromethyl group was realised by dithionite-mediated CF2C1Br-addition to the glycal double bond. Subsequently, either the chlorodifluoromethylated glyeosyl bromide (13) or the corresponding pyraneses (14 and 16) were isolated. The latter were still acetylated to the 1-O-acetyl derivatives 15 and 17, respectively. An x-ray analysis is given for the 5-O-tosylate 8.     

A super-secondary structure predicted to be common to several alpha-1,4-D-glucan-cleaving enzymes.

Predictions of protein secondary structure are used with amino acid sequence alignments to show that the N-terminal domains of cyclodextrin glucanotransferases and a yeast alpha-glucosidase may have the same super-secondary structure as alpha-amylases, i.e. an (alpha/beta)8-barrel fold. Sequence similarities provide evidence that glucanotransferases, and possibly the glucosidase, are, like alpha-amylases, Ca2+-containing enzymes. The relationship between substrate specificity and the nature of the amino acid residues proposed at the active site is discussed for the transferases and alpha-glucosidase. A set of three programs for an Apple IIe computer to carry out the calculations described by Garnier, Osguthorpe & Robson [(1978) J. Mol. Biol. 120, 97-120] and a set of four programs for an Apple IIe computer to carry out the calculations described by Levin, Robson & Garnier [(1986) FEBS Lett. 205, 303-308] have been deposited as Supplementary Publication SUP 50149 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.     

Multivariate spectrochemical analysis of interactions of three common Isatin derivatives to calf thymus DNA in vitro

Interactions of Isatin and its derivatives, Isatin-3-isonicotinylhydrazone (IINH) and Isatin-β-thiosemicarbazone (IBT), with calf thymus DNA (ctDNA) have been investigated to delineate pharmaceutical-physicochemical properties using UV–Vis/fluorescence/circular dichroism (CD) spectroscopy, viscosity measurements, and multivariate chemometrics. IINH and IBT molecules intercalate between base pairs of DNA, hypochromism in UV absorptions, increase in the CD positive band, sharp increase in specific viscosity, and the displacement of the methylene blue and Neutral Red dye in complexes with ctDNA, by the IINH and IBT molecules, respectively. The observed intrinsic binding constants (K_{b[IBT–ctDNA]?}=?1.03 × 10⁵ and K_{b[IINH–ctDNA]?}=?1.09 × 10⁵ L mol^?1) were roughly comparable to other intercalators. In contrast, Isatin binds with ctDNA via groove mode (K_{b[Isatin–ctDNA]?}=?7.32 × 10⁴ L mol^?1) without any significant enhancement in ctDNA viscosity. The fluorescence quenching of Isatin by ctDNA was observed as static. CD spectra indicated that Isatin effectively absorbs into grooves of ctDNA, leading to transition from B to C form. Thermodynamic parameters like enthalpy changes (?H < 0) and entropy changes (?S > 0) were calculated according to Van’t Hoff’s equation, indicating the spontaneous interactions. The common soft/hard chemometric methods were used not only to resolve pure concentration and spectral profiles of components using the acquired spectra but also to calculate Stern–Volmer quenching constants, binding stoichiometry, apparent binding constants (K_a), binding constants (K_b), and thermodynamic parameters. The K_b values for Isatin, IINH, and IBT were calculated as 9.18 × 10³, 1.53 × 10⁵, and 2.45 × 10⁴ L mol^?1, respectively. The results obtained from experimental-spectroscopic analyses showed acceptable agreement with chemometric outlines.     

A common protein binds to two silencers 5'' to the human beta-globin gene.

The temporal sequence of expression of human globin genes during development suggests precise regulation of these genes. Recent studies have characterized a number of DNA sequences within or flanking the human beta-globin gene which are important in its regulation and several proteins which bind to these sequences have been identified. We have found two proteins which bind 5' to the human beta-globin gene. One of these proteins, which we designate BP1, binds to two sequences, one between -550 and -527 bp relative to the cap site, the other between -302 and -294 bp. A second protein, BP2, binds to sequences between -275 and -263 bp. The binding sites for both BP1 and BP2 are in two regions which function as silencers in a transient expression assay using the human erythroleukemia cell line K562. These results and others presented here suggest that BP1 may act as a repressor protein. Negative regulation seems to be an important component of tissue and developmental specific globin gene regulation.     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains.

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.     

A database of protein structure families with common folding motifs.

The availability of fast and robust algorithms for protein structure comparison provides an opportunity to produce a database of three-dimensional comparisons, called families of structurally similar proteins (FSSP). The database currently contains an extended structural family for each of 154 representative (below 30% sequence identity) protein chains. Each data set contains: the search structure; all its relatives with 70-30% sequence identity, aligned structurally; and all other proteins from the representative set that contain substructures significantly similar to the search structure. Very close relatives (above 70% sequence identity) rarely have significant structural differences and are excluded. The alignments of remote relatives are the result of pairwise all-against-all structural comparisons in the set of 154 representative protein chains. The comparisons were carried out with each of three novel automatic algorithms that cover different aspects of protein structure similarity. The user of the database has the choice between strict rigid-body comparisons and comparisons that take into account interdomain motion or geometrical distortions; and, between comparisons that require strictly sequential ordering of segments and comparisons, which allow altered topology of loop connections or chain reversals. The data sets report the structurally equivalent residues in the form of a multiple alignment and as a list of matching fragments to facilitate inspection by three-dimensional graphics. If substructures are ignored, the result is a database of structure alignments of full-length proteins, including those in the twilight zone of sequence similarity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A three dimensional visualisation approach to protein heavy-atom structure reconstruction

Background

A commonly recurring problem in structural protein studies, is the determination of all heavy atom positions from the knowledge of the central α-carbon coordinates.

Results

We employ advances in virtual reality to address the problem. The outcome is a 3D visualisation based technique where all the heavy backbone and side chain atoms are treated on equal footing, in terms of the C_α coordinates. Each heavy atom is visualised on the surfaces of a different two-sphere, that is centered at another heavy backbone and side chain atoms. In particular, the rotamers are visible as clusters, that display a clear and strong dependence on the underlying backbone secondary structure.

Conclusions

We demonstrate that there is a clear interdependence between rotameric states and secondary structure. Our method easily detects those atoms in a crystallographic protein structure which are either outliers or have been likely misplaced, possibly due to radiation damage. Our approach forms a basis for the development of a new generation, visualization based side chain construction, validation and refinement tools. The heavy atom positions are identified in a manner which accounts for the secondary structure environment, leading to improved accuracy.

     

A proposed common structure of substrates bound to mitochondrial processing peptidase

Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha- and beta-subunits, specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Structural information of the substrate bound to MPP was obtained using fluorescence resonance energy transfer (FRET) measurement. A series of the peptide substrates, which have distal arginine residues required for effective cleavage at positions -7, -10, -14, and -17 from the cleavage site, were synthesized and covalently labeled with 7-diethyl aminocoumarin-3-carboxylic acid at the N termini and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) at position +4, as fluorescent donor and acceptor, respectively. When the peptides were bound to MPP, substantially the same distances were obtained between the two probes, irrespective of the length of the intervening sequence between the two probes. When 7-diethylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin was introduced into a single cysteine residue in beta-MPP as a donor and IANBD was coupled either at the N terminus or the +4 position of the peptide substrate as an acceptor, intermolecular FRET measurements also demonstrated that distances of the donor-acceptor pair were essentially the same among the peptides with different lengths of intervening sequences. The results indicate that the N-terminal portion and the portion around the cleavage site of the presequence interact with specific sites in the MPP molecule, irrespective of the length of the intervening sequence between the two portions, suggesting the structure of the intervening sequence is flexible when bound to the MPP.     

The structure of actinidin at 5-5 A resolution.

The structure of actinidin, a sulphydryl protease obtained from the fruit of Actinidia chinensis, has been determined from X-ray crystallographic data to a resolution of 5.5 Å. Three isomorphous heavy atom derivatives, prepared with uranyl acetate, dichloroethylenediamine platinum(II) and potassium iodomercurate(II), were used in the phase calculation, giving a mean figure of merit of 0.88. The molecule can be described as an oblate ellipsoid with approximate dimensions 50 Å × 40 Å × 36 Å, and consists of two globular units separated by a shallow cleft. Binding studies with mercuric chloride reveal two sites of attachment, both within this cleft, and although both sites are of low occupancy it is probable that one or other marks the position of the active sulphydryl group. Although the folding of the polypeptide chain of actinidin cannot be followed with certainty, it appears to be closely similar to that of papain, suggesting that these are members of a family of homologous proteins.