DNA sequestration and transcription in the oriC region of Escherichia coli

Abstract

SummaryIn Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation. The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E. coli PC2 dnaC2(ts) aligned for initiation of chromosome replication. In both seqA ¹ and dam ¹ cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered. After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA þ dam þ cells than in seqA ¹ or dam ¹ cells, but before the release of oriC from sequestration. Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA. These findings, plus analyses of transcript levels in steady-state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC. Accordingly, a proposal for the sequence of events during the interinitiation interval in E. coli is presented based on the evident coupling of transcription to replication.

Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

“…There was a second burst of repA expression at around 30 Ϯ 5 min after the shift to 30°C. Because the generation time under these conditions was about 1 h, a second round of plasmid replication most likely precedes the second burst if the plasmid goes through two rounds of replication in one generation like the PC2 chromosome (16,18). During this period, the copy number increased a little more than over 2-fold.…”

“…To follow the relationship of DNA replication and repA expression, we synchronized replication initiation by using a dnaC(ts) host, PC2 (16). In a dnaC(ts) mutant at the nonpermissive temperature (42°C), ongoing rounds of replication are allowed to complete but initiation of new rounds of replication is blocked.…”

“…In a dnaC(ts) mutant at the nonpermissive temperature (42°C), ongoing rounds of replication are allowed to complete but initiation of new rounds of replication is blocked. When incubated at 42°C, although initiation cannot happen, initiation potential builds up above normal so that on temperature shift-down, a second round of initiation follows prematurely (16,18). An exponentially growing culture of PC2 harboring a miniP1 plasmid, pSP102, was subjected to temperature shifts.…”

“…For S1 assay, probes for the repA and rpoA (control) messages were 65 and 55 nt long, respectively. They included a 20-nt long nonhomologous 3Ј tail that is selectively degraded by S1 from hybridized probes (16). This way the hybridized probes were distinguished from the residual unhybridized probes that escaped S1 digestion.…”

“…The repA probe covered P1 coordinates 775-820. The rpoA probe was as described (16). Oligonucleotides were 5Ј end-labeled and 1 ng of the repA probe and 0.1 ng of the rpoA probe were used for hybridization to 20 g of total cellular RNA and subsequently treated with S1 as recommended (Ambion).…”