Vitaly Epshtein

A Ratchet Mechanism of Transcription Elongation and Its Control

RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription.

The riboswitch-mediated control of sulfur metabolism in bacteria

Many operons in Gram-positive bacteria that are involved in methionine (Met) and cysteine (Cys) biosynthesis possess an evolutionarily conserved regulatory leader sequence (S-box) that positively controls these genes in response to methionine starvation. Here, we demonstrate that a feed-back regulation mechanism utilizes S-adenosyl-methionine as an effector. S-adenosyl-methionine directly and specifically binds to the nascent S-box RNA, causing an intrinsic terminator to form and interrupt transcription prematurely. The S-box leader RNA thus expands the family of newly discovered riboswitches, i.e., natural regulatory RNA aptamers that seem to sense small molecules ranging from amino acid derivatives to vitamins.

Linking RNA Polymerase Backtracking to Genome Instability in E. coli

Frequent codirectional 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, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional 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 avoid 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.

Transcription through the roadblocks: the role of RNA polymerase cooperation

During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins. Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro. Here we demonstrate that Escherichia coli RNAP can effectively read through the site‐specific DNA‐binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter. The anti‐roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes. These results support a cooperation model of transcription whereby RNAP molecules behave as ‘partners’ helping one another to traverse intrinsic and extrinsic obstacles.

An allosteric mechanism of Rho-dependent transcription termination

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, 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; that is, 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 allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and 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.

UvrD facilitates DNA repair by pulling RNA polymerase backwards

UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.

Riboswitch control of Rho-dependent transcription termination

Riboswitches are RNA sensors that regulate gene expression upon binding specific metabolites or ions. Bacterial riboswitches control gene expression primarily by promoting intrinsic transcription termination or by inhibiting translation initiation. We now report a third general mechanism of riboswitch action: governing the ability of the RNA-dependent helicase Rho to terminate transcription. We establish that Rho promotes transcription termination in the Mg2+-sensing mgtA riboswitch from Salmonella enterica serovar Typhimurium and the flavin mononucleotide-sensing ribB riboswitch from Escherichia coli when the corresponding riboswitch ligands are present. The Rho-specific inhibitor bicyclomycin enabled transcription of the coding regions at these two loci in bacteria experiencing repressing concentrations of the riboswitch ligands in vivo. A mutation in the mgtA leader that favors the “high Mg2+” conformation of the riboswitch promoted Rho-dependent transcription termination in vivo and in vitro and enhanced the ability of the RNA to stimulate Rhos ATPase activity in vitro. These effects were overcome by mutations in a C-rich region of the mRNA that is alternately folded at high and low Mg2+, suggesting a role for this region in regulating the activity of Rho. Our results reveal a potentially widespread mode of gene regulation whereby riboswitches dictate whether a protein effector can interact with the transcription machinery to prematurely terminate transcription.

ppGpp couples transcription to DNA repair in E. coli

A starvation survival signal fights DNA damage The alarmone guanosine-3′,5′-(bis)pyrophosphate (ppGpp) shuts down transcription in bacteria that are starving. This “stringent response” helps them conserve energy and survive adverse conditions. Kamarthapu et al. show that ppGpp is also essential for DNA repair. ppGpp couples transcription elongation to the nucleotide excision repair pathway. ppGpp helps backtrack the RNA polymerase away from the DNA damage to facilitate repair. Through inhibiting DNA replication, it also avoids dangerous collisions between the replication fork and backtracked RNA polymerase. Science, this issue p. 993 The bacterial alarmone ppGpp promotes DNA repair by shunting the transcription machinery away from DNA damage. The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

Tagetitoxin Inhibits RNA Polymerase through Trapping of the Trigger Loop

Background: Antibiotic tagetitoxin inhibits bacterial RNA polymerases (RNAPs) and RNAP III from eukaryotes. Results: We constructed a structural model of tagetitoxin bound to the transcription elongation complex. Conclusion: Tagetitoxin interacts directly with the β′ subunit trigger loop, stabilizing it in an inactive conformation. Significance: Results have implications for designing new antibiotics and understanding principles of RNAP functioning and regulation. Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the β′ subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a “slow” enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics.

Strategies and Methods of Cross-Linking of RNA Polymerase Active Center

Publisher Summary This chapter presents the basic strategies and methods of cross-linking of RNA polymerase (RNAP) active center. In order to achieve the high selectivity of affinity labeling for RNA polymerase, it is required to take advantage of “catalytic competence.” This phenomenon reflects the ability of a substrate residue cross-linked at the active center of an enzyme to convert to a cross-linked product by the same enzyme molecule according to the normal mechanism of catalysis. At the first stage RNAP is treated in the binary complex with a promoter by affinity reagent, which is an analog of initiating substrate. This results in the cross-linking of affinity reagent residues both inside and outside the active center. At the second stage the modified enzyme is supplemented with the second radioactive substrate complementary to the next base of DNA template. The chapter discusses RNA–protein cross-linking in the active center of initiating and elongation. Single-Hit degradation of polypeptides at particular residues is also discussed.. Multiple cross-linking sites can be revealed by quantitative analysis of single-hit degradation products. Another approach that appears to be very helpful for the mapping is based on the usage of functionally active enzymes assembled from the fragments of RNAP subunits.