Konstantin Brodolin

Specification of a DNA replication origin by a transcription complex

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns.

The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP). Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.

HoxB domain induction silences DNA replication origins in the locus and specifies a single origin at its boundary

In multicellular organisms, changes in the DNA replication programme could act to integrate differentiation with cell division in various developmental and transcriptional contexts. Here, we have addressed the use of DNA replication origins during differentiation in the HoxB domain—a cluster of nine genes developmentally regulated in a collinear manner. In undifferentiated mouse P19 cells, we detected several DNA replication origins in the 100 kb HoxB locus, indicating a relaxed origin use when the locus is transcriptionally silent. By contrast, in retinoic‐acid‐induced differentiated cells, when HoxB transcription is activated, a general silencing of DNA replication origins occurs in the locus except one located downstream of Hoxb1, at the 3′ boundary of the HoxB domain. Silencing of the replication origins is associated with histone hyperacetylation, whereas the active Hoxb1 origin persists as a hypoacetylated island. These findings provide direct evidence for the differentiated use of origins in HoxB genes, and we suggest that this regulation might contribute to the regulated expression of HoxB genes during development.

The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site

Worldwide spreading of drug‐resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the σ70 subunit region 3.2 and the RNAP β′ subunit switch‐2 element, which controls the clamping of promoter DNA in the RNAP active‐site cleft. Lpm abolishes isomerization of the ‘closed’‐promoter complex to the transcriptionally competent ‘open’ complex and blocks σ70‐stimulated RNA synthesis on promoter‐less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA‐strand fitting into the RNAP active‐site cleft directed by the β′ subunit switch‐2 element and the σ70 subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

In Xenopus egg extracts, DNA replication initiates preferentially at or near asymmetric AT sequences.

ABSTRACT Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using λ DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or thymine asymmetric tracks used as preferential initiation sites. In agreement with these data, AT-rich asymmetric sequences added as competitors preferentially recruit origin recognition complexes and inhibit sperm chromatin replication by increasing interorigin spacing. We also show that the assembly of a transcription complex favors origin activity at the corresponding site without necessarily eliminating the other origins. Thus, although Xenopus eggs have the ability to replicate any kind of DNA, AT-rich domains or transcription factors favor the selection of DNA replication origins without increasing the overall efficiency of DNA synthesis. These results suggest that asymmetric AT-rich regions might be default elements that favor the selection of a DNA replication origin in a transcriptionally silent complex, whereas other epigenetic elements linked to the organization of domains for transcription may have further evolved over this basal layer of regulation.

Mycobacterium tuberculosis RbpA protein is a new type of transcriptional activator that stabilizes the σA-containing RNA polymerase holoenzyme

RbpA is an RNA polymerase (RNAP)-binding protein whose presence increases the tolerance levels of Mycobacteria to the first-line anti-tuberculosis drug rifampicin by an unknown mechanism. Here, we show that the role of Mycobacterium tuberculosis RbpA in resistance is indirect because it does not affect the sensitivity of RNAP to rifampicin while it stimulates transcription controlled by the housekeeping σA-factor. The transcription regulated by the stress-related σF was not affected by RbpA. The binding site of RbpA maps to the RNAP β subunit Sandwich-Barrel Hybrid Motif, which has not previously been described as an activator target and does not overlap the rifampicin binding site. Our data suggest that RbpA modifies the structure of the core RNAP, increases its affinity for σA and facilitates the assembly of the transcriptionally competent promoter complexes. We propose that RbpA is an essential partner which advantages σA competitiveness for core RNAP binding with respect to the alternative σ factors. The RbpA-driven stimulation of the housekeeping gene expression may help Mycobacteria to tolerate high rifampicin levels and to adapt to the stress conditions during infection.

RNA Polymerase Pausing during Initial Transcription

Summary In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Mycobacterium RbpA cooperates with the stress-response σB subunit of RNA polymerase in promoter DNA unwinding

RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σA subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σA and stress-response σB, but not the alternative σ subunits from the groups 3 and 4. In contrast to σA, the σB subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

Abstract Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

Antibiotics trapping transcription initiation intermediates: To melt or to bend, what's first?

Promoter DNA melting, culminating in the loading of the single-stranded DNA template into the RNA polymerase active site, is a key step in transcription initiation. Recently, the first transcription inhibitors found to block distinct steps of promoter melting were characterized. Here, the impact of these studies is discussed with respect to the current models of transcription initiation.