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Michel Crépin, Jean-Claude Lelong and François Gros

ABSTRACT

The aim of this study is to investigate how formation of a translation initiation complex affects the rate of transcription from a phage DNA template (λ plac or Ø 80 dlac DNA). Addition of "native" 30S ribosomes to a Ø 80 dlac in vitro transcription system (including limiting amounts of purified E. coli RNA polymerase and the requisite substrates) markedly enhances the rate of RNA synthesis. Factor free, 1 M NH4Cl washed 30S or 70S ribosomes show a weak albeit detectable activity, "run off 70S" or washed 50S being inefficient. Single addition of purified initiation factor IF3 greatly magnifies (about 2–3 fold) the stimulation obtained with washed 30S subunits, the effect being catalytic with respect to IF3; IF2 addition causes a weaker and stoichiometrical effect. Maximum stimulation (up to 6 fold) is achieved by the combined addition of washed ribosomes (30S or 70S), IF1, IF2, IF3 plus the highly purified fMet-tRNAf Met species. Under such conditions, very efficient initiator tRNA binding to nascent RNA does occur. Initiation factors show no activity in the absence of ribosomes. Kasugamycin greatly reduces the stimulation of RNA synthesis in the presence of the various translation elements.

This system provides a new and very sensitive means to study the various factor dependent ribosome-messenger interactions even in the absence of initiator tRNA, thereby enabling one to analyze early translation initiation steps.

Both the frequency of transcription initiation and the rate of RNA chain propagation appear to be enhanced when RNA synthesis and initiation of protein synthesis are coupled.

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Lucie Coppin, Amandine Ferrière, Michel Crépin, Magalie Haissaguerre, Miriam Ladsous, Antoine Tabarin and Marie-Françoise Odou