Mechanisms of Transcription-Replication Collisions in Bacteria

Abstract

While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.In bacteria, DNA replication and transcription continue throughout the life cycle. The speed of the replication fork progression in Escherichia coli is ϳ1,000 bp per s (16), while the elongation rate for RNA polymerase is just 50 nucleotides (nt) per s (15); i.e., replication is approximately 20-fold faster than transcription. Since the two processes proceed simultaneously, frequent collisions between replication and transcription seem unavoidable (3, 38). Given that both processes are polar, the collisions can occur either head-on or codirectionally (Fig. 1). In the case of head-on collisions, the front edge of RNA polymerase meets the hexameric DNA helicase DnaB that moves along the lagging strand template. In the codirectional case, by contrast, the front edge of the leading strand DNA polymerase collides with the rear edge of RNA polymerase.While it is unclear a priori which type of collision is more damaging for DNA metabolism, the data on the organization of bacterial genomes point to selection against head-on collisions. Sequencing of the E. coli genome revealed that there is a bias towards codirectional alignment of transcription units with replication (2). Most strikingly, all seven ribosomal operons face the direction of their replichores. For other genes, however, this bias is much less pronounced: ϳ62% of tRNA genes and ϳ55% of protein-coding genes are aligned codirectionally with replication. Similar principles of gene arrangement were observed for other bacteria, such as Bacillus subtilis, Borrelia burgdorferi, Treponema pallidum, Haemophilus influenzae, Helicobacter pylori, Mycoplasma genitalium, and Mycoplasma pneumoniae (36), as well as for bacteriophages T7 and lambda (3).Experimental data on transcription-replication collision are relatively scarce. This problem was addressed in vitro by studying interactions between bacterial RNA polymerases and phage repl...

Archaeal Minichromosome Maintenance (MCM) Helicase Can Unwind DNA Bound by Archaeal Histones and Transcription Factors

“…This is consistent with the report that the bacteriophage T4 replication machinery paused transiently in head-on collisions with a transcription complex, although pausing was not observed when the collision was co-directional (26). Head-on collisions of the E. coli DNA replication and transcription machineries have been reported to reduce replication fork movement in vivo (27,28). All seven rRNA operons in the E. coli genome are oriented such that transcription and replication occur in the same direction, presumably to facilitate disassembly of transcription complexes during replication.…”

Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication

“…This effect has been attributed to an enhanced replication block when the transcription machinery is directed toward an oncoming replication fork. Such transcription-dependent replication pause/stall sites have been observed in yeast [17,18] as well as in bacteria [19]. In addition, in E. coli, it has been observed that the manner in which a given DNA lesion is dealt with depends on the direction of replication [20].…”

Genome-wide coorientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis

“…Paused replication forks can lead to double-strand breaks (41,43,44) and there is evidence of increased mutagenesis in the local region of doublestrand breaks (45). Essential genes are greatly enriched among transcripts cooriented with replication (20,21), probably because cells are more sensitive to mutations in essential genes and it is important to avoid disruption of their replication by head-on transcription (11). Highly expressed genes are also enriched among the cooriented transcripts (7), probably because transcription from these genes would disrupt replication more frequently when oriented opposite to replication.…”

“…Plasmid DNA replication in E. coli can also be hindered by head-on transcription from a strong inducible promoter, probably because of collisions between the replisome and RNAP (ref. 11 and references therein). Head-on transcription of tRNA genes in eukaryotes can also cause pausing of replication (12).…”