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Biomedical subjects

P Schickor

Publications and source records attributed to P Schickor.

9 recordsLinked to original sources
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↗
PubMed1993

Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase.

We have studied the early steps in RNA synthesis. The kinetic behaviour of the nascent RNA, having chain lengths between 3 and 11 bases, and the transcription fidelity were analysed using the bacteriophage T7 A1 promoter. By moving the stop-inducing base at position +12 in the wild-type template in single base steps upstream, a set of closely related templates was constructed which allowed stalling of the complexes in the registers 11, 10, 9 and 8. Using this set of templates sigma-factor release was determined. It occurs when RNA synthesis has proceeded to base position +9. Analysis of the RNA synthesis both with and without heparin yielded the following results: there are three kinds of complexes, (a) the well-known abortively transcribing complex, which is present until the RNA has reached a length of 5 bases, (b) an intermediate complex having RNA chain lengths between 6 and 8 bases, which is stably bound but has high forward as well as back reaction rates, (c) complexes with RNA chains consisting of more than 8 bases, which are stably bound and do not contain the sigma-factor. In general, the likelihood of chain elongation and the stability of the complexes increases with increasing RNA chain length in the early stages of RNA synthesis. Also the transcription fidelity increases correspondingly. Lack of fidelity leads to additional RNA products during the abortive state of transcription. "Read through" of RNA polymerase at stall positions of +8 to +11 also result from misincorporation.

Base Sequence↗
PubMed1991

Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase.

Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.

DNA, Bacterial↗
PubMed1990

Topography of intermediates in transcription initiation of E.coli.

Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.

DNA, Viral↗
PubMed1989

A cinematographic view of Escherichia coli RNA polymerase translocation.

A series of RNA synthesizing transcription complexes, initiated at the T7 A1 promoter and halted at specific base positions ranging from +12 to +40, were analyzed by footprinting techniques; exonuclease III was used to determine the position of the bound RNA polymerase on the DNA and hydroxyl radicals were used to visualize the protein--DNA contact sites within the protected areas. In the binding (open) complex without RNA there are two DNA-domains, differing in their protection pattern. The first, extending from position +18 to -13, termed 'melting domain', is fully protected, whereas the second, extending from -14 to -55, termed 'recognition domain', shows only partial protection. At this domain, RNA polymerase is attached to one side of the DNA only, as indicated by the 10-bp periodicity of the protection pattern. Our data show that the formation of a mature RNA transcribing complex is characterized by dissociation of the RNA polymerase from the recognition domain, whereby the size of the melting domain remains constant. This process is accomplished if the nascent RNA has reached a length of 11 bases. As the RNA reaches a length of 20 bases, the size of the melting domain decreases from approximately 30 to 23 bp. Further RNA synthesis leaves the protection pattern essentially unchanged. These data demonstrate that the formation of a mature RNA transcribing complex can be described by at least two transitions.

Base Sequence↗