Biomedical subjects

T Hermann

Publications and source records attributed to T Hermann.

At least 55 records · Page 3Linked to original sources

PubMed1998

RNA as a drug target: chemical, modelling, and evolutionary tools.

Dramatic technical progress in RNA synthesis and structure determination has allowed several difficulties inherent to the preparation, handling and structural analysis of RNA to be overcome, and this has led to a wealth of information about RNA structure and its relationship with biological function. It is now fully recognized that RNA molecules intervene at all stages of cell life, not only because of key sequence motifs but also because of intricate three-dimensional folds. This realization has promoted RNA to a potential therapeutic target. As in protein motifs recognizing nucleic acids, groups of the molecule interacting with RNA contribute to specific binding through defined hydrogen bonds and van der Waals docking, while other parts contribute to the driving force of binding via less specific electrostatic interactions accompanied by water and ion displacement.

Aminoglycosides↗

PubMed1998

Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme.

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metal-ion-binding pockets created by the RNA fold's electrostatic field.

Aminoglycosides↗

PubMed1997

Isoalloxazine derivatives promote photocleavage of natural RNAs at G.U base pairs embedded within helices.

We have recently shown that isoalloxazine derivatives are able to photocleave RNA specifically at G.U base pairs embedded within a helical stack. The reaction involves the selective molecular recognition of G.U base pairs by the isoalloxazine ring and the removal of one nucleoside downstream of the uracil residue. Divalent metal ions are absolutely required for cleavage. Here we extend our studies to complex natural RNA molecules with known secondary and tertiary structures, such as tRNAs and a group I intron (td). G.U pairs were cleaved in accordance with the phylogenetically and experimentally derived secondary and tertiary structures. Tandem G.U pairs or certain G.U pairs located at a helix extremity were not affected. These new cleavage data, together with the RNA crystal structure, allowed us to perform molecular dynamics simulations to provide a structural basis for the observed specificity. We present a stable structural model for the ternary complex of the G. U-containing helical stack, the isoalloxazine molecule and a metal ion. This model provides significant new insight into several aspects of the cleavage phenomenon, mechanism and specificity for G. U pairs. Our study shows that in large natural RNAs a secondary structure motif made of an unusual base pair can be recognized and cleaved with high specificity by a low molecular weight molecule. This photocleavage reaction thus opens up the possibility of probing the accessibility of G.U base pairs, which are endowed with specific structural and functional roles in numerous structured and catalytic RNAs and interactions of RNA with proteins, in folded RNAs.

Base Composition↗

PubMed1997

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

Base Sequence↗

PubMed1997

Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA.

Binding Sites↗

PubMed1997

A three-dimensional working model for a guide RNA from Trypanosoma brucei.

RNA editing in protozoan parasites is a mitochondrial RNA processing reaction in which exclusively uridylate residues are inserted into, and less frequently deleted from, pre-mRNAs. Molecules central to the process are so-called guide RNAs (gRNAs) which function as templates in the reaction. For a detailed molecular understanding of the mechanism of the editing process knowledge of structural features of gRNAs will be essential. Here we report on a computer-assisted molecular modelling approach to construct the first three-dimensional gRNA model for gND7-506, a ND7-specific gRNA from Trypanosoma brucei. The modelling process relied on chemical modification and enzymatic probing data and was validated by in vitro mutagenesis experiments. The model predicts a reasonably compact structure, where two stem/loop secondary structure elements are brought into close proximity by a triple A tertiary interaction, forming a core element within the centre of the molecule. The model further suggests that the surface of the gRNA is primarily made up of the sugar-phoshate backbone. On the basis of the model, footprinting experiments of gND7-506 in a complex with the gRNA binding protein gBP21 could successfully be interpreted and provide a first picture for the assembly of gRNAs within a ribonucleoprotein complex.

Animals↗

PubMed1997

The guide RNA database.

The RNA editing process in protozoan parasites is controlled by small RNA molecules known as guide RNAs (gRNAs). The gRNA database is a comprehensive compilation of published guide RNA sequences from eight different kinetoplastid organisms. In addition to the RNA primary sequences, information on the gene localization, the experimental verification of the transcripts, and literature citations are provided. Accessory information includes the secondary structures of fourTrypanosoma bruceigRNAs as well as a computer modelled three dimensional gRNA structure. The database is made available as a hypertext document accessible via the World Wide Web (WWW) or from the authors in a printed form.

Animals↗

PubMed1996

Strained template under the thumbs. How reverse transcriptase of human immunodeficiency virus type 1 moves along its template.

In retroviruses, such as human immunodeficiency virus type 1 (HIV-1), the reverse transcriptase (RT) copies single-stranded viral RNA into complementary DNA, which is then used as a template for synthesis of the second DNA strand. The resulting double-stranded DNA is integrated into the host genome. How RT translocates on the different templates is the subject of this study. We have developed a theoretical model for RT translocation during processive DNA synthesis. The model is based on the assumption that there are two template-binding sites, namely the helix clamps, located in the thumb subdomains of RT subunits p66 and p51. Flexibility of the p66 thumb provides undisrupted template-binding during polymerase translocation. Coordinated association and dissociation of the template at the thumbs, triggered by nucleotide incorporation, is assumed, which ensures template contact with at least one subdomain throughout translocation. We suggest that coordination between the sites is effected by stress in the template region located between the thumbs. Translocation of HIV-1 RT proceeds continuously but with different processivities on RNA and DNA templates. These findings are explained in detail by the proposed model.

Binding Sites↗

PubMed1996

The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.

Adipocytes↗

PubMed1996

Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum.

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.

Journal Article↗

PubMed1995

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Base Sequence↗

PubMed1995

Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens.

We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Animals↗

PubMed1995

Determination of nucleotide distances in RNA by means of copper phenanthroline-generated hydroxyl radical cleavage pattern.

In contrast to the commonly used Fe(II)-EDTA, bis(orthophenanthroline)-copper(I) (OP-Cu) first generates hydroxyl radicals after binding to RNA. Due to diffusion, the hydroxyl radicals can cleave neighboring nucleotides in a distance r of up to 1.5 nm to the OP-Cu binding site. Using the known structure of tRNAPhe as a reference, we show that the hydroxyl radical cleavage pattern generated by a specifically bound OP-Cu shows a 1/r dependence on the distance of the cleaved nucleotide to the OP-Cu binding site. We propose that OP-Cu is a suitable probe for obtaining data on the distances between nucleotides in RNA, which can be used in modeling the structure of the examined RNA. However, this information is restricted to about three to four bases surrounding an OP-Cu binding site.

Anticodon↗

PubMed1994

The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding.

Algorithms↗

PubMed1993

Hydroxyl radical footprint analysis of human immunodeficiency virus reverse transcriptase-template.primer complexes.

Human immunodeficiency virus type 1 reverse transcriptase protects sugar moieties of a model template.primer DNA in a region from positions +3 to -15 from hydroxyl radical attack. A protected region of equivalent size migrates in concert with the translocating enzyme, as shown by hydroxyl radical footprints of replication complexes after primer extension by 4, 10, and 19 nt. The pattern of these footprints suggests that the DNA template.primer is in the A conformation when complexed with reverse transcriptase. Enhanced accessibility of the DNA template strand around position -15 to hydroxyl radicals indicates a conformational change in the template induced by the C-terminal RNase H-containing domain of p66 reverse transcriptase.

Base Sequence↗

PubMed1993

Formation of retinoid X receptor homodimers leads to repression of T3 response: hormonal cross talk by ligand-induced squelching.

Thyroid hormone receptors (TRs) form heterodimers with retinoid X receptors (RXRs). Heterodimerization is required for efficient TR DNA binding to most response elements and transcriptional activation by thyroid hormone. RXRs also function as auxiliary proteins for several other receptors. In addition, RXR alpha can be induced by specific ligands to form homodimers. Here we report that RXR-specific retinoids that induce RXR homodimers are effective repressors of the T3 response. We provide evidence that this repression by RXR-specific ligands occurs by sequestering of RXR from TR-RXR heterodimers into RXR homodimers. This ligand-induced squelching may represent an important mechanism by which RXR-specific retinoids and 9-cis retinoic acid mediate hormonal cross talk among a subfamily of nuclear receptors activated by structurally unrelated ligands.

Animals↗

PubMed1993

V-erbA requires auxiliary proteins for dominant negative activity.

The avian v-erbA protein is an important example of a dominant negative oncogene. It has been identified as a highly mutated form of its cellular homolog, the thyroid hormone receptor alpha (TR-alpha), and its biological activity has been correlated with its repressor function on certain receptor-regulated genes. Although v-erbA has lost the hormone responsiveness of its cellular homolog, it has retained DNA-binding activity, and it has been implied that this function is required for repression and transformation. Here we demonstrate that v-erbA forms heterodimers with the retinoid X receptor (RXR-alpha). Only heteromeric v-erbA-RXR-alpha complexes show DNA-binding strong enough to account for its potent repressor function. In addition, v-erbA-RXR-alpha heterodimers specifically bind natural thyroid hormone-responsive elements (TREs) but not retinoic acid-responsive elements (RAREs). Repression of TRE-controlled gene expression by v-erbA requires the presence of RXR-alpha with the natural TREs tested. In contrast, natural RAREs investigated here do not bind the v-erbA-RXR-alpha heterodimer and also are not significantly repressed by v-erbA. Carboxy-terminal mutations that abolish v-erbA-RXR-alpha heterodimer formation also abolish v-erbA repressor activity. These data suggest that interaction of v-erbA with RXRs or similar auxiliary receptors is essential for the dominant negative activity of the v-erbA oncogene.

Base Sequence↗

PubMed1992

Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid.

Retinoid response pathways are mediated by two classes of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A central question is whether distinct response pathways are regulated by these two classes of receptors. The observation that the stereoisomer 9-cis-retinoic acid binds with high affinity to RXRs suggested that this retinoid has a distinct role in controlling RXR activity, but it was almost simultaneously discovered that RXRs function as auxiliary receptors for RARs and related receptors, and are essential for DNA binding and function of those receptors. Hence, although RARs seem to operate effectively only as heterodimeric RAR/RXR complexes, RXRs themselves apparently function predominantly, if not exclusively, as auxiliary receptors. Here we report that 9-cis-retinoic acid induces RXR homodimer formation. Our results demonstrate a new mechanism for retinoid action by which a ligand-induced homodimer mediates a distinct retinoid response pathway.

Animals↗