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Biomedical subjects

K H Scheit

Publications and source records attributed to K H Scheit.

At least 91 records · Page 5Linked to original sources
PubMed1981

The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli.

Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA. These reactions were carried out by E. coli RNA polymerase on T7 DNA in the presence of rifampicin. The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined. On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds. The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not.

Adenine Nucleotides↗
PubMed1981

Cell-free expression of the beta-galactosidase gene: a model system to study the effects of nucleotide analogs on transcription-translation.

A cell-free system for the expression of the beta-galactosidase gene was employed to study the effects of the UTP and CTP analogs: s2UTP, s2CTP, f5UTP and rTTP on transcription-translation. From the analogs investigated, only rTTP turned out to be able to substitute UTP in the cell-free synthesis of beta-galactosidase. In case of the other analogs listed above, the incorporation of even a small fraction of analog into rRNA resulted in drastic inhibition of beta-galactosidase synthesis.

Escherichia coli↗
PubMed1981

Substrate specificity of CTP-synthetase from E. coli.

The substrate specificity of CTP-synthetase from E. coli was investigated by means of UTP analogs. This study revealed that the three main structural elements of the UTP molecule were important for the substrate specificity of the enzyme. CTP-synthetase seems to possess an absolute requirement for the beta-D-ribose 5-triphosphate part in UTP. Substitutents in 5-position of UTP, exceeding the size of a tritium atom abolish substrate function.

Carbon-Nitrogen Ligases↗
PubMed1978

Primed abortive initiation of RNA synthesis by E. coli RNA polymerase on T7 DNA. Steady state kinetic studies.

Ternary complexes of T7 DNA, RNA polymerase and the antibiotic rifampicin carry out the promoter specific abortive initiation when dinucleoside monophosphates were employed as primers. Primed abortive initiation, leading to synthesis of trinucleoside diphosphates, only occured with combinations of primers and substrates complementary to a promoter region of 8 base pairs centered around the origin of transcription. The steady state kinetics of three abortive initiations at T7 promoter A3 were studied in detail. The reactions appeared to be truly ordered. Affinity constants, maximal velocities and elementary step rate constants were thus obtained. The stimulation by dinucleoside monophosphate primers is brought about by positively effecting the function of the substrate site rather then by their higher affinity to the primer site of the transcriptional complex.

Base Sequence↗
PubMed1977

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.

Affinity Labels↗
PubMed1977

The stereochemical basis of template function.

The behavior of nucelotides with thioketo-substituted pyrimidine bases (4-thiouracil, 2-thiouracil and 2-thiocytosine) or amino-analogue purine bases (2-aminopurine and 2,6-diaminopurine) in transcription and translation was investigated. The experimental results obtained led to the following conclusions. 1. The stereochemical basis of substrate selection in transcription is the geometry of Watson-Crick base pairs A-U (or A-T) and G-C between substrate and template bases. 2. The topology of the active site of Escherichia coli RNA polymerase is precisely adopted to the geometry of Watson-Crick base pairs. 3. The enzyme active site discriminates between A-U (A-T) and G-C base pairs. An essential feature in this discrimination is the 6-NH2 group of the A-U (A-T) base pair and the 2-keto group of cytosine in the G-C base pair. 4. The codon properties of a nucleic acid base in messenger RNA can be predicted on the basis of its specificity in polynucleotide interactions. There seems to be no evidence for the participation of protein topological sites in the control of the specificity of codon-anticodon interactions in translation.

Animals↗
PubMed1977

Steady state kinetic studies of initiation of RNA synthesis on T7 DNA in the presence of rifampicin.

The steady state kinetics of initiation of T7 DNA transcription by RNA polymerase holo enzyme from E. coli in the presence of rifampicin and the two substrates ATP and UTP were studied. Under these conditions, the enzyme catalyzes exclusively the promotor specific synthesis of pppApU. The kinetic data are in agreement with the mechanism of a truly ordered reaction. Binding of the initiating nucleotide ATP to the transcriptional complex occurs prior to the binding of the substrate UTP. Release of pppApU is most probably the rate limitinig step. Km constants were found to be 0.6 mM for ATP and 0.31 mM for UTP, respectively. The substrate inhibition pattern indicated that the substrate site exhibits a finite affinity for incorrect nucleoside triphosphate (Ki = 2.3 mM). A similar non specific binding to the 3-OH site could not be demonstrated.

Adenosine Triphosphate↗
PubMed1976

Dissociation kinetics of complexes between the antibiotic rifamycin and DNA-dependent RNA polymerase from Escherichia coli.

Phosphocellulose chromatography was employed to measure the binding of 3-(2-[14C]acetamidoethyl)-thiorifamycin(abbreviated[14C]AcNHEtS-Rif) to RNA polymerase core enzyme. The technique yielded a correct value for the stoichiometry of interaction. The same method was successfully applied to an investigation of the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex. We observed biphasic dissociation kinetics not in agreement with the existence of one single first-order dissociation step. Assuming two independent dissociation reactions, rate constants have been evaluated differing roughly by 10-fold (1.7 X 10(-3) s-1 and 1.5 X 10(-4) s-1 at 25 degrees C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data are interpreted as indirect evidence for the existence of two enzyme species differing in their dynamic properties with respect to the binding of the antibiotic rifamycin. Our data furthermore lend support to the assumption that the two enzyme forms are in equilibrium. The observed sigmoidal dependence of the dissociation rate on temperature could indicate a conformational transition of RNA core polymerase with a transition midpoint at around 20 degrees C. The dye, rose bengal, was found to be as effective as AcNHEtS-Rif itself as a competing agent. The dissociation kinetics of the [14C]-AcNHEtS-Rif - core-polymerase complex in the presence of an excess of the dye rose bengal were found to be very similar to those measured in the presence of AcNHEtS-Rif. This could mean that rose bengal and AcNHEtS-Rif compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS-Rif - core-polymerase - poly[d(A-T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow first-order kinetics. The dissociation rate constant was calculated to be 1.7 X 10(-4) s-1. Experiments to determine the effect of the antibiotic streptolydigin on the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex have not led to conclusive results.

Binding Sites↗
PubMed1976

The effect of thioketo substitution on uracil-2-aminopurine and uracil-2, 6-diaminopurine interactions in polynucleotides.

The existence of the complexes poly[r(s4U)] . poly[r(n2h6A] and poly-[r(s4U)] . poly[r(n2A)] was demonstrated by means of spectrophotometric titration and sedimentation veolicty analysis. According to the absorption-temperature profiles thioketo substitution of poly[r(U)] . poly[r(n2h6A)] led to stabilisation of the helical structure, thus implying that the 4-thioketo group does not participate in s4U . n2h6A base pairing. In the case of poly[r(s4U)] . poly[r(n2A)] drastic destabilistaion of the helical structure by thioketo substitution was observed. This indicates that the thioketo substituents participate in s4U .n2A base pairing.

Binding Sites↗
PubMed1976

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).

Animals↗
PubMed1975

The modification of DNA-dependent RNA polymerase from Escherichia coli by an alkylating derivative of rifamycin SV.

3-(2-Bromo[1-14C]acetamidoethyl)-thio-rifamycin SV, abbreviated BrAcNEtS-Rif, and alkylating derivative of rifamycin SV was synthesized. A four-fold excess of BrAcNEtS-Rif inhibited the enzymic activity of RNA polymerase from Escherichia coli to 97%. Incubation of RNA polymerase with Br[14C]AcNEtS-Rif led to covalent substitution. The reaction of Br[14C]AcNEtS-Rif with enzyme at a ratio of 1.4:1 and a concentration of 63 nM was found to proceed with a half life of 1 h at 37 degrees C. The enzyme could be protected from reaction with BrAcNEtS-Rif by either rifampicin or the hybrid [poly(dT)]-[r(Ap)5a]. The modification of holoenzyme by Br[14C]AcNEtS-Rif in the presence of p-hydroxymercuribenzene sulfonic acid (pOH-HgBzSO3H) or 4 M LiCl occurred with faster kinetics and led to a higher degree of substitution. Reaction of Br[14C]AcNEtS-Rif with RNA polymerase core enzyme caused predominant substitution of subunit beta. In the case of RNA polymerase holenzyme the radioactive substituents were evenly distributed between subunits beta and sigma. Apparently the topology of the rifamycin binding site of holoenzyme, similarly to core enzyme, precludes attacks of nucleophilic functions from beta' and alpha, but it allows nucleophilic functions from subunits beta and sigma to react with equal probability on BrAcNEtS-Rif. In the presence of a 20-fold excess of pOH-HgBzSO3H, the modification of holoenzyme was drastically altered. Virtually all substitution took place on subunit beta', very little on beta and none on subunits sigma and alpha.

Alkylating Agents↗