Annie Schwartz

The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post‐transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho‐dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U‐rich transcript region directly upstream from the terminator. Antitermination is modulated by trans‐acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

A stepwise 2'-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase.

The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho–RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large (∼7-nucleotide-long) discrete steps triggered by 2′-hydroxyl activation events. This periodic 2′-OH–dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

Apoptosis induction and DNA interstrand cross-link formation by cytotoxic trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)]: Cross-linking between d(G) and complementary d(C) within oligonucleotide duplexes

We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross‐linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans‐[PtCl2(NH3)2] (trans‐DDP) by dimethylamine and isopropylamine. The data show that trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is able to circumvent resistance to cis‐[PtCl2(NH3)2] (cis‐DDP, cisplatin) in A2780cisR cells. In fact, trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] shows a cytotoxic potency higher than that of cis‐DDP and trans‐DDP, with the mean IC50 values being 11, 58, and 300 μM, respectively. In addition, at equitoxic doses (concentrations of the platinum drugs equal to their IC50 values) and after 24 hours of drug treatment, the level of induction of apoptosis by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is twice that produced by cis‐DDP. Under the same experimental conditions, trans‐DDP does not induce significant levels of apoptosis in A2780cisR cells. After 24 hours of incubation of A2780cisR cells at concentrations equal to the IC50 value of the platinum drugs, the level of DNA interstrand cross‐links (ICLs) induced by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is two and three times higher, respectively, than those induced by cis‐DDP and trans‐DDP. We also found that trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] formed DNA ICLs between guanine and complementary cytosine. We propose that, in A2780cisR cells, the induction of apoptosis by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is related to its greater ability (relative to cis‐DDP and trans‐DDP) to form DNA ICLs.

Transcription Termination Factor Rho Can Displace Streptavidin from Biotinylated RNA

In Escherichia coli, binding of the hexameric Rho protein to naked C-rich Rut (Rho utilization) regions of nascent RNA transcripts initiates Rho-dependent termination of transcription. Although the ring-shaped Rho factor exhibits in vitro RNA-dependent ATPase and directional RNA-DNA helicase activities, the actual molecular mechanisms used by Rho to disrupt the intricate network of interactions that cement the ternary transcription complex remain elusive. Here, we show that Rho is a molecular motor that can apply significant disruptive forces on heterologous nucleoprotein assemblies such as streptavidin bound to biotinylated RNA molecules. ATP-dependent disruption of the biotin-streptavidin interaction demonstrates that Rho is not mechanistically limited to the melting of nucleic acid base pairs within molecular complexes and confirms that specific interactions with the roadblock target are not required for Rho to operate properly. We also show that Rho-induced streptavidin displacement depends significantly on the identity of the biotinylated transcript as well as on the position, nature, and length of the biotin link to the RNA chain. Altogether, our data are consistent with a “snow plough” type of mechanism of action whereby an early rearrangement of the Rho-substrate complex (activation) is rate-limiting, physical force (pulling) is exerted on the RNA chain by residues of the central Rho channel, and removal of structural obstacles from the RNA track stems from their nonspecific steric exclusion from the hexamer central hole. In this context, a simple model for the regulation of Rho-dependent termination based on the modulation of disruptive dynamic loading by secondary factors is proposed.

The functional anatomy of an intrinsic transcription terminator

To induce dissociation of the transcription elongation complex, a typical intrinsic terminator forms a G·C‐rich hairpin structure upstream from a U‐rich run of approximately eight nucleotides that define the transcript 3′ end. Here, we have adapted the nucleotide analog interference mapping (NAIM) approach to identify the critical RNA atoms and functional groups of an intrinsic terminator during transcription with T7 RNA polymerase. The results show that discrete components within the lower half of the hairpin stem form transient termination‐specific contacts with the RNA polymerase. Moreover, disruption of interactions with backbone components of the transcript region hybridized to the DNA template favors termination. Importantly, comparative NAIM of termination events occurring at consecutive positions revealed overlapping but distinct sets of functionally important residues. Altogether, the data identify a collection of RNA terminator components, interactions and spacing constraints that govern efficient transcript release. The results also suggest specific architectural rearrangements of the transcription complex that may participate in allosteric control of intrinsic transcription termination.

Simple Enzymatic Assays for the In Vitro Motor Activity of Transcription Termination Factor Rho from Escherichia coli

The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA-DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rhos motor activity in a more direct and quantitative manner.

Transplatin-modified Oligonucleotides as Potential Antitumor Drugs

The discovery by Rosenberg and co-workers (1,2) of the induction of filamentous growth in bacteria cells by platinum-amine complexes has been the starting point of much work devoted to the chemistry of these complexes, their binding to biomolecules (particularly to DNA), and their biologic activity. One major achievement is the successful use of cis-diamminedichloroplatinum(II) (cisplatin) in the treatment of several human cancers. Cisplatin triggers cell death by apoptosis, but the complete mechanism of action of the drug and the development of resistance have not yet been elucidated (3–5). Cellular DNA is the target of cisplatin through covalent interaction (6–8). The lesions formed in the reaction between DNA and cisplatin have been identified in vitro and in vivo (mainly intrastrand and interstrand crosslinks), as well as the distortions they induce in the DNA double helix (9,10). Recently, several studies have demonstrated that proteins that bend DNA also interact specifically with cisplatin-modified DNA at d(GpG) and d(ApG) sites (11–19). This gives strong support to the hypothesis that the major 1,2-intrastrand crosslinks between adjacent purine nucleotides play a key role, although one cannot discard the role of the interstrand crosslinks.

ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

Comparison Between poly(dG-dC)·poly(dG-dC) and DNA Modified by cis-diamminedichloroplatinum (II): Immunological and Spectroscopic Studies

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.

Detection of minor adducts in cisplatin‐modified DNA by transcription footprinting

Two DNA restriction fragments containing either a d(GC)5 or a d(TTGCTTGATTAGTTGTGTT) insert were subjected to reaction with cis‐diamminedichloroplatinum(II) and were then used as templates for RNA synthesis by T7 RNA polymerase. Within the d(GC)5 insert, interstrand cross‐links are preferentially formed. Within the second insert, the reactivity order of the potential binding sites is d(ApG) > d(GpC/ GpC) = d(GpA) > d(GpTpG). In the presence of cyanide ions, the adducts are much less stable at the d(GpA) sites than at the d(GpCpG) sites, in double‐stranded DNA.