Étude du RNA synthétisé in vitro

Abstract

Studies on RNA synthesized in vitro The bulk of RNA synthesized by purified E. coli RNA polymerase on calf thymus and T 7 DNA as templates, has been chromatographed on methylated serum albumin column. The elution pattern is very similar to those obtained with pulse labelled RNA from cells. The RNA's obtained in vitro are eluted, as three main fractions, namely II, III and IV, located respectively before the 16-S and 23-S RNA and after the 23-S RNA. RNA's from Fractions II and III are not aggregates made of either different RNA chains or RNA and carrier rRNA, as demonstrated by the following data: (1) Fractions II and III are present even if the carriers rRNA are omitted. (2) Thermal or mechanical degradation of the RNA lowers Fraction IV, increases Fractions II and III and induces the formation of new peaks. (3) The elongation of RNA can be limited by incorporation of cordycepin triphosphate. Under these conditions, Fraction IV disappears and Fractions II and III lose their sharpness. (4) The RNA length can be directly measured by the ratio 3H belonging to the chain nucleotides, to 32P attached to the initiator nucleotide. The evolution of this ratio through the pattern is coherent and the relation between the position of a given RNA in the elution pattern and its length, is perfectly reproducible. After 20 min synthesis, the lengths of RNA's constitutive of Fractions II, III and IV, are respectively: 1350–1500, 2700–2900 and 5000–5500 nucleotides. When the synthesis of RNA is allowed to proceed after a short pulse with a labelled nucleotide followed by a chase, the evolution of the pattern of fractions shows that the RNA of Fractions II and III are precursors of the RNA IV which are also growing. In fact, when a synchronized elongation of the RNA chains is allowed to proceed, we obtain only Fraction IV. These data, toegether with the fact that no nuclease activity has ever been detected in our preparations, show that the particular heterogeneity of the RNA is a genuine property of the material synthesized in vitro. The relative quantities of Fractions II, III and IV vary with the DNA, RNA polymerase and nucleotide concentrations. The kinetics of appearance of the different fractions and the knowledge of their respective lengths show that the average rate of RNA synthesis decreases with time of incubation. It is 90–100 nucleotides/sec during the first 30 sec and 20–27 nucleotides/sec during the first 3 min. One explains the presence of these specific intermediary fractions by the fact that the rate of synthesis is not homogeneous along the entire DNA molecule, and that the initiation of RNA chains is not instantaneous. The kinetics of the synchronized elongation of the RNA chains shows 4 plateaux during the first 3 min, even though each plateau does not represent an intermediary fraction of defined chain length. It appears nevertheless that two kinds of phenomena, one rapid, one slow, are involved in the elongation of the RNA chains.