The influence of potassium and rifampicin on the expression of bacteriophage T2 prereplicative genes in vitro and in vivo

Abstract

The transcription of specific mRNA from T2 phage DNA by purified Escherichia coli RNA polymerase was markedly affected by the concentration of KCl. Thus the genes for dihydrofolate reductase and deoxynucleotide kinase were transcribed at 50 m m KCl, but at 200 m m KCl the synthesis of the kinase mRNA was nearly eliminated and that of the reductase was increased slightly. Rifampicin-resistant initiation complexes formed at 200 m m KCl failed to produce appreciable kinase mRNA despite reduction of the KCl concentration to 50 m m following initiation of RNA chain elongation. This result suggests that the RNA polymerase holoenzyme does not bind to deoxynucleotide kinase promoters in the presence of 200 m m KCl in a manner capable of initiating synthesis of this message. Initiation complex formation at 50 m m KCl, however, permitted the synthesis of deoxynucleotide kinase mRNA at 200 m m KCl indicating that once formed the initiation complex is capable of producing message even at high salt. The KCl effect on gene expression was reproduced in a coupled E. coli cell-free system directed by T2 DNA. However, in this case the inhibition of protein synthesis at high concentrations of KCl was due primarily to the chloride ions and could be alleviated by substituting acetate for chloride. The synthesis of mRNA in the coupled system was similar with either anion. Analysis of proteins synthesized in the coupled system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, like deoxynucleotide kinase, the synthesis of several additional gene products was influenced by the concentration of KCl. The genes coding for these products, including deoxynucleotide kinase, belong to a class whose transcription is reduced or eliminated in vivo on addition of rifampicin at 1 min postinfection and are referred to as “quasilate.” Evidence is presented to indicate that the mRNAs for deoxynucleotide kinase, dihydrofolate reductase, and deoxycytidylate deaminase are produced in excess of the levels needed for optimal synthesis of these enzymes during normal infection.