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Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions., Competing Interests: The authors declare no conflict of interest.

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity ( K d ∼ 10 -14 -10 -17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays., Competing Interests: Conflict of interest statement: The CL7/Im7 purification technology is protected by a PCT patent (PCT/US2016/065843) filed by the UAB Research Foundation.

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

Bacterial Gre transcript cleavage factors stimulate the intrinsic endonucleolytic activity of RNA polymerase (RNAP) to rescue stalled transcription complexes. They bind to RNAP and extend their coiled-coil (CC) domains to the catalytic centre through the secondary channel. Three existing models for the Gre-RNAP complex postulate congruent mechanisms of Gre-assisted catalysis, while offering conflicting views of the Gre-RNAP interactions. Here, we report the GreB structure of Escherichia coli. The GreB monomers form a triangle with the tip of the amino-terminal CC of one molecule trapped within the hydrophobic cavity of the carboxy-terminal domain of a second molecule. This arrangement suggests an analogous model for recruitment to RNAP. Indeed, the beta'-subunit CC located at the rim of the secondary channel has conserved hydrophobic residues at its tip. We show that substitutions of these residues and those in the GreB C-terminal domain cavity confer defects in GreB activity and binding to RNAP, and present a plausible model for the RNAP-GreB complex.

The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5'-[(alpha,beta)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active ('insertion') site closed through refolding of the trigger loop (TL) into two alpha-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive ('preinsertion') substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the beta barrel of NusG. To our knowledge, such an all-beta to all-alpha transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the beta' subunit coiled coil, the major target site for the initiation sigma factors.

The mechanism of pre-protein export through the bacterial cytoplasmic membrane, in which the SecA ATPase plays a crucial role as an "energy supplier", is poorly understood. In particular, biochemical and structural studies provide contradictory data as to the oligomeric state of SecA when it is integrated into the active trans-membrane translocase. Here, we report the 2.8 A resolution crystal structure of the Thermus thermophilus SecA protein (TtSecA). Whereas the structure of the TtSecA monomer closely resembles that from other bacteria, the oligomeric state of TtSecA is strikingly distinct. In contrast to the antiparallel (head-to-tail) dimerization reported previously for the other bacterial systems, TtSecA forms parallel (head-to-head) dimers that are reminiscent of open scissors. The dimer interface is abundant in bulky Arg and Lys side-chains from both subunits, which stack on one another to form an unusual "basic zipper" that is highly conserved, as revealed by homology modeling and sequence analysis. The basic zipper is sealed on both ends by two pairs of the salt bridges formed between the basic side-chains from the zipper and two invariant acidic residues. The organization of the dimers, in which the two pre-protein binding domains are located proximal to each other at the tip of the "scissors", might allow a concerted mode of substrate recognition while the opening/closing of the scissors might facilitate translocation.

The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6(1)22 or P6(5)22, with unit-cell parameters a = b = 45.46, c = 599.93 A. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 A, alpha = 80.62, beta = 75.37, gamma = 75.41 degrees ). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 A resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress.

The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3(1(2))21 (a = b = 168.6, c = 149.8 A) and P6(1(5))22 (a = b = 130.9, c = 564.6 A). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.

Gre factors enhance the intrinsic endonucleolytic activity of RNA polymerase to rescue arrested transcription complexes and are thought to confer the high fidelity and processivity of RNA synthesis. The Gre factors insert the extended alpha-helical coiled-coil domains into the RNA polymerase secondary channel to position two invariant acidic residues at the coiled-coil tip near the active site to stabilize the catalytic metal ion. Gfh1, a GreA homolog from Thermus thermophilus, inhibits rather than activates RNA cleavage. Here we report the structure of the T. thermophilus Gfh1 at 2.4 A resolution revealing a two-domain architecture closely resembling that of GreA. However, the interdomain orientation is strikingly distinct (approximately 162 degrees rotation) between the two proteins. In contrast to GreA, which has two acidic residues on a well fixed self-stabilized alpha-turn, the tip of the Gfh1 coiled-coil is flexible and contains four acidic residues. This difference is likely the key to the Gre functional diversity, while Gfh1 inhibits exo- and endonucleolytic cleavage, RNA synthesis, and pyrophosphorolysis, GreA enhances only the endonucleolytic cleavage. We propose that Gfh1 acidic residues stabilize the RNA polymerase active center in a catalytically inactive configuration through Mg2+-mediated interactions. The excess of the acidic residues and inherent flexibility of the coiled-coil tip might allow Gfh1 to adjust its activity to structurally distinct substrates, thereby inhibiting diverse catalytic reactions of RNA polymerase.

The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 148, c = 115.2 A and a = b = 59.3, c = 218.9 A, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 A resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 A of the Thermus thermophilus RNA polymerase (RNAP)-Tgt complex revealed that the Tgt-binding site within the RNAP secondary channel overlaps that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the beta and beta' residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg(2+) ion, which is distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg(2+) ion has a key role in stabilization of an inactive transcription intermediate. Remodeling of the active site by metal ions could be a common theme in the regulation of catalysis by nucleic acid enzymes.

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

The Escherichia coli gene encoding a regulator of stringent response and virulence, DksA, which contains a canonical Zn finger motif, was cloned and expressed, and the purified protein was crystallized by the hanging-drop vapor-diffusion technique in two different space groups, P2(1)2(1)2(1) (a = 91.32, b = 96.59, c = 117.48 A) and C222 (a = 80.6, b = 115.1, c = 149.57 A). The crystals belonging to space group P2(1)2(1)2(1), improved by macroseeding, diffract beyond 2.0 A at a synchrotron. Three complete atomic resolution multiple anomalous dispersion diffraction data sets were collected from the same crystal of the P2(1)2(1)2(1) crystal form at the absorption edge for Zn atoms.

Bacterial transcription is regulated by the alarmone ppGpp, which binds near the catalytic site of RNA polymerase (RNAP) and modulates its activity. We show that the DksA protein is a crucial component of ppGpp-dependent regulation. The 2.0 A resolution structure of Escherichia coli DksA reveals a globular domain and a coiled coil with two highly conserved Asp residues at its tip that is reminiscent of the transcript cleavage factor GreA. This structural similarity suggests that DksA coiled coil protrudes into the RNAP secondary channel to coordinate a ppGpp bound Mg2+ ion with the Asp residues, thereby stabilizing the ppGpp-RNAP complex. Biochemical analysis demonstrates that DksA affects transcript elongation, albeit differently from GreA; augments ppGpp effects on initiation; and binds directly to RNAP, positioning the Asp residues near the active site. Substitution of these residues eliminates the synergy between DksA and ppGpp. Thus, the secondary channel emerges as a common regulatory entrance for transcription factors.

Guanosine-tetraphosphate (ppGpp) is a major regulator of stringent control, an adaptive response of bacteria to amino acid starvation. The 2.7 A resolution structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to the same site near the active center in both independent RNAP molecules in the crystal but in strikingly distinct orientations. Binding is symmetrical with respect to the two diphosphates of ppGpp and is relaxed with respect to the orientation of the nucleotide base. Different modes of ppGpp binding are coupled with asymmetry of the active site configurations. The results suggest that base pairing of ppGpp with cytosines in the nontemplate DNA strand might be an essential component of transcription control by ppGpp. We present experimental evidence highlighting the importance of base-specific contacts between ppGpp and specific cytosine residues during both transcription initiation and elongation.

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.

The gene encoding LysX, an essential component of the lysine-biosynthesis pathway in Thermus thermophilus (molecular weight approximately equal 31,000 Da), was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups, C2 (unit-cell parameters a = 124.7, b = 51.4, c = 103.6 A, beta = 122.8 degrees ) and R3 (a = b = 122.6, c = 97.6 A). Crystals improved by macroseeding diffracted to beyond 2.3 and 3 A resolution for the C2 and R3 crystal forms, respectively. Complete diffraction data sets were collected for the C2 and R3 crystal forms at 2.5 and 3.1 A resolution, respectively. Crystals of selenomethionine-containing LysX protein were obtained by cross-microseeding, using the native microcrystals as a seed. Structure determination is now in progress.

RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (alpha(2), beta, beta', omega and sigma(70)) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 A resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P3(2), with unit-cell parameters a = b = 236.35, c = 249.04 A, and have perfect twinning along the threefold axis. A complete data set at 3 A resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the sigma(70) subunit is now in progress.

In bacteria, the binding of a single protein, the initiation factor sigma, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A resolution. In the structure, two amino-terminal domains of the sigma subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of sigma is near the outlet of the RNA-exit channel, about 57 A from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.