Open AccessAffinité différentielle de la RNA polymérase pour divers polyribonucléotides synthétiques
Author(s) -
Hirschbein L.,
Dubert J.M.,
Babinet C.
Publication year - 1967
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1967.tb00054.x
Subject(s) - transcription (linguistics) , dna , polymerase , enzyme , rna , chemistry , microbiology and biotechnology , rna polymerase , base pair , stereochemistry , biochemistry , biology , gene , philosophy , linguistics
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 K m 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.