In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase

Abstract

Plasmids pNF1337 and pNF1341, which contain part of the L10 operon including the RNA polymerase beta-subunit gene, have been used as templates in vitro to investigate expression of the beta-subunit gene. For these studies, the synthesis of the first dipeptide of the beta subunit, fMet-Val, was measured instead of that of the entire protein. By using this dipeptide system, we studied the effects of RNA polymerase holoenzyme and L factor (nus A gene product) on fMET-Val synthesis and compared the relative effects of the primary and secondary promoters in the L10 operon on expression of the beta-subunit gene. The results show that the inhibitory effect of RNA polymerase on beta-subunit synthesis and the stimulatory effect of L factor occur before formation of the first dipeptide bond. In this in vitro system, the secondary promoters account for about 50% of the total fMet-Val synthesized. Although the primary promoter is sensitive to guanosine 5'-diphosphate 3'-diphosphate in vitro, the secondary promoters are not affected by this nucleotide.