Initiation de chaînes de RNA par la RNA polymérase in vitro

In the presence of cordycepin triphosphate (3′‐deoxyadenosine triphosphate), an analogue of ATP, RNA polymerase synthesizes very short RNA chains in vitro. These chains, labelled on the first nucleotide with [γ32P]GTP, are acid soluble but are retained on nitrocellulose filters. After alkaline hydrolysis, chromatography on Dowex 1 (CI−)shows that the 32P‐labelled molecules behave like guanosine tetraphosphate. When filtered on Sephadex G‐75 the oligonucleotides synthesized are associated with a structure of high molecular weight, which is probably the complex DNA‐enzyme, and remain so after ribonucleases or deoxyribonuclease treatment. The number of nascent oligonucleotides measured by incorporation of [γ32P]GTP, represents only 50% of the chains initiated by guanosine in the control. This observation, together with the fact that nascent oligonucleotides are not sensitive to pancreatic or T1 ribonuclease, shows that they are involved in a structure which determines their stability. This structure could be an Enzyme‐(DNA‐RNA hybrid) complex at the growing point of the RNA chains. The kinetics of initiation of RNA are the same whether chain propagation is prevented or not. The number of RNA chains initiated per phage T7 DNA molecule has been measured with [γ32P]ATP and [γ32P]GTP. It increase linearly with the amount of RNA polymerase added although the amount of RNA made tends towards a maximum. On the other hand, the number of nascent oligonucleotides made does not increase with an excess of enzyme, which shows the existence of a finite number of initiation sites. These results are thought to indicate that initiation sites are reutilized during normal RNA synthesis. The number of chains initiated by adenine and guanine per phage T7 DNA at the point where RNA synthesis, as a function of RNA polymerase concentration, departs from proportionality, is around 50.