Secondary Structure Model Research Articles

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing.

The genes encoding pea and potato mitochondrial tRNAGly and pea mitochondrial tRNASer(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNAGly gene sequences revealed A7:C66 mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNAGly cDNA clones showed that these mispairings are not corrected by C66 to U66 conversions, as observed in plant mitochondrial tRNAPhe. Likewise, a U6:C67 mismatch identified in the acceptor stem of the pea tRNASer(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNACys. In vitro processing reactions with the respective tRNAGly and tRNASer(GCU) precursors show that such conversions are not necessary for 5' and 3' end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.

Structural analysis of the P10/11-P12 RNA domain of yeast RNase P RNA and its interaction with magnesium.

The P10/11-P12 RNA domain of yeast RNase P contains several highly conserved nucleotides within a conserved secondary structure. This RNA domain is essential for enzyme function in vivo, where it has a demonstrated role in divalent cation utilization. To better understand the function of this domain, its structure and alterations in response to magnesium have been investigated in vitro. A secondary structure model of the P10/11-P12 RNA domain had been previously developed by phylogenetic analysis. Computer modeling and energy minimization were applied to the Saccharomyces cerevisiae P10/11-P12 domain to explore alternatives and additional interactions not predicted by the phylogenetic consensus. The working secondary structure models were challenged with data obtained from 1H NMR and in vitro chemical and enzymatic probing experiments. The solution structure of the isolated domain was found to conform to the phylogenetic prediction within the context of the holoenzyme. Structure probing data also discriminated among additional base contacts predicted by energy minimization. The withdrawal of magnesium does not appear to cause gross refolding or rearrangement of the RNA domain structure. Instead, subtle changes occur in the solution accessibility of specific nucleotide positions. Most of the conserved nucleotides reported to be involved in magnesium utilization in vivo also display magnesium-dependent changes in vitro.

Studies on the large subunit rRNA genes and their flanking regions of leuconostocs

The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris were amplified by polymerase chain reactions and sequenced. The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp. dextranicum were also sequenced. The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions. A single tRNAAla gene without the 3'-terminal CCA sequence was found in spacer-1 regions. Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs. For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing. Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers. Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents. Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures. Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs. We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA - spacer-1 - tRNAAla - 23S rRNA - spacer-2 - 5S rRNA-3'.Key words: leuconostocs, rrn operons, rDNA, 23S, spacer regions, polymerase chain reaction.

Multiple functions of the transcribed spacers in ribosomal RNA operons.

rRNA operons contain about 25% transcribed spacer sequences in addition to the 16S, 23S, 5S and tRNA genes. The spacer sequences are removed from the primary rRNA transcript by a series of co-ordinated nucleolytic events. Besides the role in rRNA processing, the spacer sequences are also involved in transcription and the ribosome assembly. In this study we analyze the spacer between tRNA and 23S rRNA genes. Based on computer modeling and chemical probing data, a model for the transient secondary structure of the intergenic spacer is proposed. Mutational analysis has shown that the transient secondary structure around the 5' end of 23S rRNA is involved in ribosome assembly. We propose that the transient structure at the 5' end of 23S rRNA directs 23S rRNA folding into the mature structure and facilitates ribosomal large subunit assembly.

Modeling studies of conformational changes in the substrate-binding loop in Drosophila alcohol dehydrogenase.

Three-dimensional structures of seven short-chain dehydrogenases/reductases show that these enzymes share common structural features. Sequence alignment studies of Drosophila alcohol dehydrogenase (DADH), with an unknown 3D-structure, and four short-chain dehydrogenases/reductases with known X-ray structures suggest that DADH shares the same structural features. However, the substrate binding regions, which are located in the C-terminal region of these enzymes, share little sequence homology, because of the wide variety of substrates used. X-ray structures of short-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, in the C-terminal region, upon substrate binding. This substrate-binding loop is located between a strand and a helix and may contain one or two small helices. Secondary structure predictions and modeling studies of this substrate-binding loop in DADH predict that the two helices may also be present in this enzyme. The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-F differ in a replacement of threonine by lysine at position 192, which is located at a central position in the substrate-binding loop. The positive charge of lysine may move significantly on substrate binding, resulting in a direct charge interaction with NAD+ in the enzyme-substrate complex, explaining a very strong influence of pH on the binding of ADH-S for the NAD+ analogue Cibacron Blue. This indicates that the ADH S/F polymorphism has a direct influence on the catalytic properties of the enzyme.

The guide RNA database.

Guide RNAs (gRNAs) are small, metabolically stable RNA molecules which perform a pivotal, template-like function during the RNA editing process in kinetoplastid protozoa. The gRNA database currently contains 250 guide RNA sequences as well as secondary and tertiary structure models and other relevant information. The database is made available as a hypertext document accessible via the World Wide Web (WWW) at the URL: http://www.biochem.mpg.de/ goeringe/

The Na+/I- symporter (NIS): recent advances.

The Na+/I- symporter (NIS) catalyzes the accumulation of iodide into thyroid cells, an essential step in the biosynthesis of thyroid hormones. As a result of the isolation of the rat NIS cDNA, steadfast advances in the study of NIS at the molecular level have resulted in the following accomplishments: generation of high-affinity anti-NIS antibodies, elucidation of NIS stoichiometry and specificity by electrophysiological analysis, biochemical and immunological experimental testing of the proposed NIS secondary structure model, monitoring the regulation of NIS protein expression by thyroid stimulating hormone and iodide, characterization of the rat NIS gene promoter, isolation of the cDNA clone encoding human NIS and subsequent determination of human NIS genomic organization, description of NIS mutations in patients with congenital lack of iodide transport, and the molecular identification of NIS in extrathyroidal tissues.

Database on the structure of small ribosomal subunit RNA

About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/

Sequences around the fragmentation sites of the large subunit ribosomal RNA in the family Rhizobiaceae. 23S-like rRNAs in Rhizobiaceae.

We demonstrated that the representatives of the family Rhizobiaceae possess, instead of one single 23S rRNA molecule, three different sets of 23S-like rRNA fragments with sizes of about: 135 b and 2.6 kb (set 1); 135 b, 400 b, and 2.2 kb (set 2); 135 b and two molecules of about 1.3 kb (set 3). In two of the fragmentations, intervening sequences--IVS I and IVS II--are involved. The IVS I is connected to a cleavage of the 23S rRNA primary transcript into two modules (135 b and 2.6 kb large). The IVS II is located at position 543 of the gene, and it leads to an additional processing of the 2.6 kb rRNA species into two molecules with sizes of about 400 b and 2.2 kb. In contrast to the IVS I, which is a common feature of all rhizobia, the IVS II is present in a limited number of strains only. The primary and secondary structures of the regions of the unmatured 23S rRNA transcript possessing IVS I (helix 9) and IVS II (helix 25) were analysed. On the basis of our analyses we propose secondary structure models of the parts of the matured 23S rRNA-like molecules of rhizobia corresponding to the helices 9 and 25. The third fragmentation of the rhizobial 23S rRNA represents a break in the central part of the 2.6 kb-large rRNA and it leads to an occurrence of two fragments with approximately equal size of about 1.3 kb. We have demonstrated that the central fragmentation is not connected to the presence of IVSs but probably to a minor change in the nucleotide sequence in the central part of the 2.6 rRNA.

Single substitutions of phosphorothioates in the HDV ribozyme G73 define regions necessary for optimal self-cleaving activity

Phosphorothioate (NTPalphaS) analogues were incorporated into the HDV genomic ribozyme by transcription with T7 polymerase. The introduction of a sulfur in place of the pro-Rp oxygen at the phosphate 5'to positions A64, A63, A43, U27, G62, C61, C44, C41, C22and C21appeared to inhibit self-cleavage activity of the G73 genomic ribozyme. Except for position C22, elevated levels of Mg2+rescued the reaction to various extents. When the sites were identified in the RNA sequence, they were clustered in three distinct regions that, in the secondary structure models, are predicted to be primarily single-stranded. Two of these regions have been proposed to form extensive interactions that are thought to involve a homopurine base pair. The third region is thought to be directly associated with assembly of the cleavage site.

Probing the secondary structure of MCS4 RNA of Mycoplasma capricolum.

MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum. Its function is unknown. The conformation of MCS4 RNA (125 nucleotides in length) in solution was investigated using chemical and enzymatic probes. Single and double-stranded regions were estimated by means of diethyl pyrocarbonate (DEPC) and by dimethyl sulfate (DMS) modifications. Ribonuclease V1 was also used to identify paired or stacked nucleotides. Based on these data, a secondary structure model for MCS4 RNA containing four stem structures was constructed.

Collapse transitions in protein-like lattice polymers: The effect of sequence patterns

The collapse transition of lattice protein-like heteropolymers has been studied by means of the Monte Carlo method. The protein model has been reduced to the α-carbon trace restricted to a high coordination lattice. The sequences of model heteropolymers contain two types of mers: hydrophobic/nonpolar (H) and hydrophilic/polar (P). Interactions of HH and PP pairs were assumed to be negative (weaker attractions of PP pairs) while the contact energy for HP pairs was equal to zero. All sequence-specific short-range interactions have been neglected in the present studies. It has been found that homopolymeric chains undergo a smooth collapse transition to a dense globular state. The globule lacks any signatures of local ordering that could be interpreted as a model of protein secondary structure. Hetero-polymers with the sequences of hydrophilic and hydrophobic residues characteristic for α- and β-type proteins undergo a somewhat sharper (though continuous) collapse transition to a dense globular state with elements of local ordering controlled by the sequence. The helical pattern induces more secondary structure than the β-type pattern. For all examined sequences the level of local ordering was lower than the average secondary structure content of globular proteins. The results are compared with other theoretical work and with known experimental facts. The implications for the reduced modeling of protein systems are briefly discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 537–548, 1997

Structure and function of the thyroid iodide transporter and its implications for thyroid disease

The thyroid iodide (I−) transporter, a Na+/I− symporter (NIS), is an intrinsic 618-amino-acid membrane glycoprotein that catalyzes the accumulation of I− into thyrocytes, an essential step in the biosynthesis of thyroid hormones. Remarkable progress has recently been achieved in the study of NIS at the molecular level as a result of the isolation of the cDNA encoding rat NIS. Among the main accomplishments are the isolation of the cDNA clone encoding human NIS, electrophysiological analysis and elucidation of the mechanism, stoichiometry, and specificity of rat NIS, generation of anti-rat NIS antibodies for use in NIS characterization, proposal and experimental testing of NIS secondary structure models, analysis of the regulation of NIS expression by thyroid-stimulating hormone and I−, characterization of the regulation of the rat NIS gene, elucidation of the genomic organization of human NIS, identification of an NIS mutation in a case of congenital lack of I“ transport, and detection of anti-NIS autoantibodies in thyroid autoimmune disease.

Stability and transcriptional regulation of gamma-glutamyl transpeptidase mRNA expression in the initial segment of the rat epididymis.

Gamma-glutamyl transpeptidase (GGT) is an enzyme believed to play a role in the protection of maturing spermatozoa in the epididymis. Our previous studies have shown that four GGT mRNAs (I-IV) transcribed from the single-copy rat GGT gene are differentially expressed and regulated in the rat epididymis. In particular, the normal expression of GGT mRNA(IV) in the epididymal initial segment is dependent upon the presence of testicular factors. The objective of this study was to test the hypothesis that the decreased expression of GGT mRNA(IV) in the initial segment following the in vivo removal of testicular factors by efferent duct ligation (EDL) is due to a decrease in stability and/or transcription rate. The stability of the GGT mRNAs was evaluated by measuring the rate of mRNA decay. These stability studies showed that GGT mRNA(IV) exhibited a rapid initial decay that slowed at later times to a decay rate similar to that of GGT mRNAs(II,III). The decay rates were not different following sham-operation or EDL, and thus the stability of GGT mRNAs were not influenced by the in vivo loss of testicular factors. Results of transcription analysis revealed that the transcription rate of GGT mRNA(IV) in the initial segment fell by approximately 68% following a 12-hour EDL. Additionally, secondary-structure models indicate two families of folding patterns for GGT mRNA(IV), which could be the reason for the two decay regimes detected in the stability study. Thus, the decreased expression level of GGT mRNA(IV) in the initial segment following the in vivo loss of testicular factors is a function of a decreased transcription rate and intricate decay kinetics.

Thermodynamic model of secondary structure for α‐helical peptides and proteins

Thermodynamic model of secondary structure for α‐helical peptides and proteins

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of alpha-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining alpha-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The alpha-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each alpha-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by 1H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set 1] and [set 2]) and for 30 mostly alpha-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by 1H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix-coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix-coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area.

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

Hepatitis B virus X gene 1751 to 1764 mutations: implications for HBeAg status and disease.

A translational stop in the hepatitis B virus (HBV) precore codon 28 and specific changes in the core promoter region of the X gene have been suggested to influence the level of circulating HBeAg in patients. We analysed the core promoter region and precore sequences from 59 HBV strains (including 14 from the databank) of different genotypes and from patients with different HBeAg/anti-HBe patterns. The initiator and TATA elements for transcription of precore and pregenomic RNA were highly conserved. The majority of X gene deletions in the core promoter region would lead to translational frame-shifts and stops, truncating the C-terminal end of the X protein. We found significant associations between specific changes in core promoter positions 1762 to 1764, or in precore codon 28, and absence of circulating HBeAg. For the core promoter mutations alone, this association was related to the apparent degree of liver damage (as estimated by alanine aminotransferase levels) at the time of sampling. Mutations at nucleotides 1762 and/or 1764 were often accompanied by point mutations at positions 1751 to 1755. Since mutations at nucleotide positions 1762 and 1764 have recently been shown by in vitro studies to suppress HBeAg production with a concomitant enhancement of virus production, disappearance of the HBeAg-positive phenotype associated with 1762 to 1764 mutations may thus have at least as much significance for the course of infection as HBeAg absence associated with precore codon 28 stop mutations. These observations are considered against a secondary structural model for the 3' end of HBV pregenomic RNA which also predicts enhancement of virus replication after mutation at positions 1762 and 1764.

Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody.

The Na+/I- symporter (NIS) is the plasma membrane protein that catalyzes active I- transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; approximately 65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a approximately 87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the approximately 87 kDa-polypeptide into a approximately 50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (approximately 56 kDa) and the mature approximately 87 kDa form. Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.