SUMMARY Frequent co-directional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Co-directional collisions with backtracked (arrested) ECs lead to DNA double strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to evade replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance.

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria1 -3. Although Rho is the first termination factor to be discovered 4 , the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle, i.e. it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allostericallyinduced rearrangement of the RNAP catalytic center via the evolutionary conserved mobile trigger loop (TL) domain, which is also required for EC dissociation. The key structural/functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution.Rho is the general transcription termination factor in bacteria. Concerted cycles of ATP binding, hydrolysis and release induce conformational changes that pull the nascent transcript through the cavity of the hexamer and induce Rho to translocate along the RNA in the 5′ → 3′ direction [5][6][7][8] . Classical tracking models postulate that, once loaded onto the nascent RNA, Rho catches up to the EC and pulls the transcript away from RNAP and the DNA template [9][10][11] . Extensive biochemical and structural analysis of Rho over the past two decades has explained the molecular details of RNA loading and translocation [5][6][7][8][9][10][11] , but not the actual mechanism of termination. Different models that address this issue have been proposed 9,10,12 ( Supplementary Fig. 1). The majority of these models envisioned RNAP as passive in the termination process. Indeed, simple kinetics, i.e. the speed at which RNAP elongates, is thought to be the major determinant of Rho termination sites, many of which coincide with RNAP pause sites. Although kinetic coupling is certainly an important parameter in controlling the efficiency of Rho termination 13,14 , it does not define the conformational state of the enzyme, which renders the EC susceptible to Rho.To study Rho termination in vitro we constructed two templates containing the T7A1 promoter fused to well-characterized Rho termination sequences: trpt' and aRut14 -16 . We also utilizedCorrespondence and requests for materials should be addressed to E.N. (evgeny.nudler@nyumc.org). * These authors contributed equally to this work.Author Contributions V.E., D.D., and J.W. conducted the experimental work, discussed the results, and commented on the manuscript. E.N. de...

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA – the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.

Excessive manganese exposure is toxic, but a comprehensive biochemical picture of this assault is poorly understood. Whether oxidative stress or reduced energy metabolism under manganese exposure causes toxicity is still a debate. To address this, we chose Δmnt P Escherichia coli, a highly manganese-sensitive strain, in this study. Combining microarray, proteomics, and biochemical analyses, we show that the chronic manganese exposure rewires diverse regulatory and metabolic pathways. Manganese stress affects protein and other macromolecular stability, and envelope biogenesis. Most importantly, manganese exposure disrupts both iron-sulfur cluster and heme-enzyme biogenesis by depleting cellular iron level. Therefore, the compromised function of the iron-dependent enzymes in the tricarboxylic acid cycle, and electron transport chain impede ATP synthesis, leading to severe energy deficiency. Manganese stress also evokes reactive oxygen species, inducing oxidative stress. However, suppressing oxidative stress does not improve oxidative phosphorylation and cell growth. On the contrary, iron supplementation resumed cell growth stimulating oxidative phosphorylation. Therefore, we hypothesize that affected energy metabolism is the primal cause of manganese toxicity.

The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.