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H Heumann

Publications and source records attributed to H Heumann.

At least 37 records · Page 2Linked to original sources
PubMed1997

FIS activates sequential steps during transcription initiation at a stable RNA promoter.

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.

Carrier Proteins↗
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

Influence of Mg2+ and temperature on formation of the transcription bubble.

The transcription bubble formed in the binding complex of T7A1 promoter upon Escherichia coli RNA polymerase was analyzed by chemical probes, namely by single-strand specific reagents, to map the unpaired bases in the bubble, and by FeEDTA, to analyze the accessibility of the DNA backbone. The latter probe could also be used as a local hydroxyl radical probe placed close to the Mg2+-binding site in the active center. The data show that the transcription bubble consists of two parts, an Mg2+-dependent part and an Mg2+-independent part, both having individual transition temperatures. The data further suggest that formation of a transcription active open complex is preceded by a transition state complex having enhanced affinity for those Mg2+ ions presumably participating in the formation of the catalytic site. Our data also suggests that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent. One/two of the three Mg2+ ions are responsible for the polymerization, the other two/one for enlargement of the transcription bubble.

Amino Acid Sequence↗
PubMed1997

Mammalian tRNA(Lys)3 and pre-tRNA(Lys)3 variants as primers and inhibitors of viral cDNA synthesis by HIV reverse transcriptase in vitro.

We constructed a set of mammalian tRNA(Lys)3-pseudogenes, whose pre-tRNAs are processing-deficient in HeLa extract. The 3'-flanking region was designed partially or completely complementary to the PBS-flanking nucleotides of the U5-region of HIV-1 genomic RNA. We show that only the pre-tRNA with completely hybridized 3'-flanking region is efficiently extended by the HIV-1 reverse transcriptase, whereas the partially complementary pre-tRNA(Lys)3, the 3'-terminal 10 nucleotides of which are not hybridized to the viral template, is unable to prime the cDNA-synthesis.

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

Probing the higher order structure of RNA with peroxonitrous acid.

Potassium peroxonitrite (ONOOK) and [Fe(EDTA)]2- were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]2- gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOH) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]2- is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the 'inside' of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA(Lys3) which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA(Lys3).

Animals↗
PubMed1996

Mapping of catalytic residues in the RNA polymerase active center.

When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the beta' subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutation. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex. The mutant fails to support Fe2+-induced cleavage of DNA or protein. Thus, the NAD-FDGD motif is involved in chelation of the active center Mg2+.

Amino Acid Sequence↗
PubMed1995

FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter.

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.

Base Sequence↗
PubMed1995

In vitro transcription close to the melting point of DNA: analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 degrees C using chemical probes.

The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85 degrees C. We show that the T. maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E. coli polymerase. We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing. This study revealed that the T. maritima polymerase goes through a series of isomerisation events very similar to the E. coli polymerase, i.e. the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.

2,2'-Dipyridyl↗
PubMed1995

Translocation of the Escherichia coli transcription complex observed in the registers 11 to 20: "jumping" of RNA polymerase and asymmetric expansion and contraction of the "transcription bubble".

Translocation of DNA-dependent RNA polymerase along the DNA template during RNA synthesis encompasses continuous as well as discontinuous steps. This is demonstrated by chemical probing of transcription complexes stalled in consecutive registers of RNA synthesis at base positions +11, +12, +14, +16, +18, and +20. The "transcription bubble" translocates by continuous opening of the downstream edge in tandem with the growing RNA chain and discontinuous closing at the upstream edge after at least nine steps of RNA synthesis. The position of the enzyme remains unchanged during extension of the transcription bubble and "jumps" 10 bp downstream simultaneously with collapse of the transcription bubble.

Base Sequence↗
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

Osteoinductive substances in middle ear surgery.

There have been a number of stages in the development of cholesteatoma surgery. After the introduction of the radical operation the disadvantages of this procedure led to the development of more conservative operative techniques, for example the anterior-posterior approach with preservation of the posterior wall of the external ear canal or the obliteration of the mastoid cavity with muscle flaps or various materials. The assumption that organic fragments of the bone enable certain unspecified mesenchymal cells to form bone led to this investigation. Allogenic bone matrix and gelatin were implanted in the epitympanic recess in 16 rabbits. The animals were sacrificed 2, 4, and 8 weeks after the operation and the temporal bones were examined histologically. Both the implanted substances led to the formation of new bone in the epitympanic recess in rabbits. The newly formed bone bonded itself with the bony wall of the epitympanic recess. The existence of foreign body giant cells could not be verified. Perhaps the difficulties involved in treating cholesteatomas could be solved with osteoinductive substances.

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↗
PubMed1994

The structure of the RNA polymerase-promoter complex. DNA-bending-angle by quantitative electrooptics.

The complex formed between RNA polymerase holoenzyme from Escherichia coli and the strong promoter A1 from the phage T7 has been characterized by measurements of the electric dichroism. The dichroism decay time constant of a promoter DNA fragment with 126 bp increases upon binding of the polymerase, but the increase is less than expected for simple addition of the components at the known binding site. Our results demonstrate a protein-induced decrease of the hydrodynamic DNA dimensions, which is not a consistent with an increased flexibility, but indicates bending of the double helix with a relatively narrow distribution of bending angles. We have characterized the degree of DNA bending by bead model simulations and used, in addition to our present experimental data, the available information on the overall size and shape of the RNA polymerase, together with the location of the DNA bending center at the starting point of RNA synthesis. We conclude that the bending angle is 45 degrees (+/- 5 degrees).

Bacteriophage T7↗
PubMed1994

From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases.

Multisubunit RNA polymerases in prokaryotes and eukaryotes share an evolutionarily conserved core. Here, we compare the processes of promoter recognition, transcription initiation and transcript elongation by human RNA polymerase II and by the RNA polymerase of the eubacterium Escherichia coli. Although these two polymerases have diverged widely in structure, important functions have been conserved, suggesting that the basic mechanisms of RNA transcription are similar in eukaryotes and prokaryotes.

DNA-Directed RNA Polymerases↗