Affinité différentielle de la RNA polymérase pour divers polyribonucléotides synthétiques

Abstract

Using synthetic polyribonucleotides templates as models, the problems of attachment or detachment of DNA dependent‐RNA polymerase to its template have been investigated. In addition, the hypothesis that sites of initiation or termination of transcription might be determined by some specific base sequences was tested with these models. In order to allow a distinction between the transcription process and its first step, which is the binding of the enzyme to its template, the inhibition by different polyribonucletides of the transcription of DNA was examined. A wide range of relative effects was found (Fig. 3). It is remarkable that the polyribonucleotides Poly G and Poly I showing the highest affinities for the enzyme, as well as the homopolymer pair Poly (G + C) of low affinity could not be transcribed into their complementary sequences when used as template. When using the polyribonucleotides as template instead of DNA, the Km for triphosphate binding is about 30 fold higher. It was shwon in inhibition experiments that for a given system, a polyribonucleotide was more efficient as an inhibitor as the concentration of its complementary triphosphate was increased (Fig. 4): this result shows that the triphosphate contributes to the stability of the polyribonucleotide‐enzyme complex. The properties of Poly I or Poly G of having very high affinity for RNA polymerase and of not being transcribed could be used to trap the enzyme not bound to its template at any time before or during the transcription process. Preliminary results are reported in Fig.5. The fact that Poly (G + C) has barely detectable affinity for RNA polymerase can explain the kinetic results obtained when Poly C is used as a template; the G/C ratio reaches one at the end of the reaction and no further incorporation of GMP is observed when enzyme or GTP is added. Only the addition of Poly C can allow resumption of incorporation of the complementary nucleotides (Fig.2A). Consistent with these results is the observation that the enzyme and the product of the reaction migrate separately in a sucrose gradient centrifugation. Our studies show that the relative affinity of RNA polymerase for different homopolymer pairs Poly (A + U), Poly (G + C) is dependent on the nature of the bases involved in these ordered double helical structures.

On the Binding of Rifampicin to the DNA‐Directed RNA Polymerase from Escherichia coli

“…Hence we have to assume that during chain elongation a different conformation prevails which does not allow the attachment of rifampicin. Indeed, several earlier observations of the resistance of the enzyme during chain elongation to other inhibitors such as heparin [40] or polyriboinosinic acid [41], to tryptic digestion, high salt concentrations or higher temperatures [42 -441 support this notion. Rifampicin even on prolonged incubation does not bind to the enzyme which is in the state of RNA chain elongation.…”

The Selectivity of Transcription

“…While the intrinsic rate of RNA chain initiation by RS complexes is not strongly affected by temperature or elevated ionic strengths, the fraction of RNA polymerase holoenzymc molecules in RS complexes decreases dramatically at temperatures below 20°C or at salt concentrations above 0.1 M NaC1 (176,205). Under thess ame conditions enzyme in binary complexes becomes more sensitive to attack by polyribonucleotides (116,284) or heparin (26,253). These properties of holoenzyme-DNA mixtures couid all be explained if binding failed to occur or was greatly reduced under these conditions, or if the conditions favored binding at nonspecific (Class B) sites on DNA.…”

“…polymerase is inhibited by low concentrations of a number of polyanions, including RNA homopolymers (116,179,(282)(283)(284) and the polyanions heparin (26,285) polyethanesulfonate (286). These polyanions bind tightly to the enzyme at a site sites which are blocked when the enzyme is in a ternary complex growing an RNA chain (26,(285)(286)(287).…”

Transcription of Bacteriophage T4 DNA in vitro: Selective Initiation with Dinucleotides

“…To prevent further initiation polyinosinic acid was added and the assay mixture chilled to 0 "C. This treatment releases all polymerase molecules which have not initiated. Poly(1) competes for free polymerase molecules [37,44,45]. Then the other three triphosphates were added allowing the extension of the initiated transcripts at 37 "C. For further details see Materials and Methods.…”