Rifampin-induced initiation of chromosome replication in dnaR-deficient Escherichia coli cells

Abstract

The dnaR130 mutant of Escherichia coli, which was thermosensitive in initiation of chromosome replication, was capable of thermoresistant DNA synthesis in the presence of rifampin at a low concentration that allowed almost normal RNA synthesis. The DNA synthesis in the presence of the drug depended on protein synthesis at the high temperature. The protein synthesis in the dnaR-deficient cells provided a potential for thermoresistant DNA synthesis to be induced at a high dose of the drug that almost completely prevented RNA synthesis. The induced synthesis was synchronously initiated from oriC and proceeded semiconservatively toward terC. The replication depended on the dnaA function, which was essential for normal initiation of replication from oriC. The capability for drug-induced replication was abolished by certain rifampin resistance mutations in the ␤ subunit of RNA polymerase. Thus, the drug can induce the dnaA-dependent initiation of replication in the dnaR-deficient cells through its effect on RNA polymerase. This result implies that the dnaR product is involved in the transcription obligatory for the initiation of replication of the bacterial chromosome.Replication of the Escherichia coli chromosome is initiated from a fixed site, oriC, at a specific stage of the cell cycle. The initiation of a new round of replication is sensitive to rifampin (Rif), an inhibitor of RNA synthesis, at a stage when protein synthesis is no longer required (13,19). Involvement of RNA polymerase in the initiation of replication is inferred from genetic evidence that some Rif resistance (Rif r ) mutations in rpoB, the gene for the ␤ subunit of RNA polymerase, can suppress the thermosensitivity of dnaA mutants in initiation of replication (4,5,34). In addition, some mutations in the rpoB or rpoC gene have been shown to elevate the number of chromosomes per cell (26,27,39). It was recently shown that transcription of the mioC and gidA genes adjacent to oriC occurs in a cell cycle-dependent manner (24, 40). However, the regulatory mechanism of transcription for the initiation of chromosome replication has remained unknown.It has been shown that replication of oriC plasmids is sensitive to Rif and is influenced by transcription toward or away from the oriC region (1,3,14,15,23,37,38). The transcription affecting oriC plasmid replication is modulated by the dnaA product, which binds to DnaA box sequences within or around the oriC region (1,14,15,22,28,35,37). The studies of oriC plasmid replication in vitro have revealed that transcription by RNA polymerase preceding the initiation of replication from oriC assists DnaA protein to open the DNA duplex (6, 36). The duplex opening leads to formation of prepriming complexes by DnaB and DnaC proteins and subsequent synthesis of RNA primers for DNA synthesis by DnaG primase (12). Because the action of DnaG primase is insensitive to Rif (43), the sensitivity of oriC plasmid replication to the drug is ascribed to transcriptional activation of oriC by RNA polymerase.Recently, I showed that ...

Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli

“…Although PRPP is a precursor obligatory for the biosynthesis of various nucleotides, the dnaR mutant is not defective in vital functions except for the dnaA-dependent initiation of chromosome replication (Sakakibara, 1992a). The dnaR product of the prs gene seems to participate in a transcriptional event for the initiation of replication, because the dnaR defect is suppressed by certain rifampicin-resistance rpoB mutations in RNA polymerase (Sakakibara, 1995) and by rifampicin that affects the action of RNA polymerase in the initiation of replication (Sakakibara, 1996). Some of the dnaR-suppressing rpoB mutations suppress the dnaA defect in initiation of replication (Sakakibara, 1995).…”

Use of Rifampicin in T7 RNA Polymerase-Driven Expression of a Plant Enzyme: Rifampicin Improves Yield and Assembly

“…Its further potential as a cure (26) as well as its side effects (5,17,29) are still being examined. Second, rifampicin became a tool of biochemical and genetic analysis of intracellular mechanisms of various processes (11,21,25,28). It found use in expression studies of recombinant proteins.…”

Interference of the Simian Virus 40 Origin of Replication by the Cytomegalovirus Immediate Early Gene Enhancer: Evidence for Competition of Active Regulatory Chromatin Conformation in a Single Domain

“…Activation of DNA replication by transcription has been found in both prokaryotic (3,11,35,56,70) and eukaryotic (48, 60) cells. On the other hand, transcription has been found to suppress replication in bacterial plasmid DNA (47, 53) and in yeast (77, 82), tetrahymena (61), and human (33) cells.…”