The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli

Abstract

Despite the widely accepted view that transcription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC. Furthermore, neither gid nor mioC transcription is required in cells deficient in the histone-like proteins Fis or IHF. However, oriC that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of mioC and especially gid is needed to activate oriC only under suboptimal conditions. We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within oriC, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.Initiation of replication of the Escherichia coli chromosome occurs at a unique site, oriC. Initiation depends primarily on DnaA protein, which binds to five 9-bp repeats within oriC and facilitates duplex melting at three AϩT-rich 13-mers (see Fig. 1). DnaB helicase subsequently enters the opened duplex and leads to priming and chain elongation reactions resulting in bidirectional replication. oriC is a complex regulatory region containing recognition sites for many positive and negative regulatory proteins, including Fis, IHF, SeqA, and IciA, some or all of which are thought to help precisely regulate initiations occurring at oriC (see ref. 1 for review).Early physiology experiments by Lark (2) and Messer (3) first suggested that RNA polymerase (RNAP) is somehow involved in initiation at oriC. This was inferred from findings that rifampicin (an inhibitor of RNAP) inhibits a new round of DNA synthesis at a time when protein synthesis is no longer required. Genetic evidence as well suggests a role of RNAP in initiation. Mutations in rpoB and rpoC, which encode the ␤ and ␤Ј subunits of RNAP, respectively, have been shown to increase copy numbers of both the chromosome and chimeric plasmids (oriC plasmids) carrying both an oriC site and a ColE1-type replication origin (4, 5). Further, specific rpoB mutations have been shown to suppress the temperature sensitivity phenotype of certain dnaA(Ts) mutations (6). Despite the evidence suggesting an involvement of RNAP, a specific transcription event required for initiation has not been identified.Of promoters possibly involved in transcriptional activation of initiation, one likely candidate is the promoter of gidAB. The gid promoter (P gid ) is situated just counterclockwise of oriC ( Fig. 1) and transcription proceeds leftward away from oriC. oriC plasmids in which the gid promoter has been inactivated exhibit decreased transformation efficiency and stability as well as decreased replication in vitro (7,8). The twin-domain model of Liu and Wang (9) predicts that an actively transcribing RNAP generates local domains of increased negative supercoiling behind it and positive supercoiling in front of it. It...

Relations Between Replication and Transcription

“…The proposed mechanisms by which transcription from mioC could inhibit replication were that transcription could impair formation of initiation complex at the origin via physical interactions which would displace DnaA from DNA or through the generation of local changes in the DNA topology destabilizing the initiation complex. However, while the replication time in cells lacking mioC promoter was unaffected (Bates et al 1997;Lobner-Olesen et al 1992), transcription from this promoter became essential for cell viability when the dnaA box R4 was deleted suggesting a positive role for mioC transcription in replication initiation (Bates et al 1997). Despite of all the data obtained over 3 decades, no clear consensus on the role of transcription from the mioC gene on replication has been achieved.…”

MioC Is an FMN-binding Protein That Is Essential forEscherichia coli Biotin Synthase Activity in Vitro

“…The mioC gene is located next to oriC on the E. coli chromosome and although several investigations have tried to establish a role for mioC transcription in chromosome replication (22)(23)(24), the results were inconclusive (25,26 (7); lane 5, assay containing all standard components plus fraction 25 from the final Mono Q column (see Fig. 2) .…”

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.…”

“…The CMV enhancer was also found to exhibit an inhibitory effect on the CMV enhancer-promoter driving chloramphenicol acetyltransferase expression in a dose-dependent manner. Together these results suggest that inhibition of SV40 origin-based DNA replication by the CMV enhancer is due to intramolecular competition for the formation of active chromatin structure.A majority of DNA replication origins in both prokaryotic (3,11,35,42,53,56,59) and eukaryotic (1, 5, 12, 17-19, 24, 25, 36, 38, 41, 43, 48, 60, 62-66, 68, 71, 75, 78, 81, 83, 84, 93-95) cells are closely associated with a transcription unit. Recent characterization of origins of replication in mammalian cells has shown that the origins are located very close to a transcription unit or even within a transcription unit (see review in reference 20).…”

“…A majority of DNA replication origins in both prokaryotic (3,11,35,42,53,56,59) and eukaryotic (1, 5, 12, 17-19, 24, 25, 36, 38, 41, 43, 48, 60, 62-66, 68, 71, 75, 78, 81, 83, 84, 93-95) cells are closely associated with a transcription unit. Recent characterization of origins of replication in mammalian cells has shown that the origins are located very close to a transcription unit or even within a transcription unit (see review in reference 20).…”