Marina N. Vassylyeva

Structural basis for transcription elongation by bacterial RNA polymerase.

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-Å 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 β′-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.

Structural basis for substrate loading in bacterial RNA polymerase

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches.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-Å resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-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 α-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.

Regulation through the secondary channel--structural framework for ppGpp-DksA synergism during transcription

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.

Structural Basis for Transcription Regulation by Alarmone ppGpp

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.

Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins

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.

Transcription inactivation through local refolding of the RNA polymerase structure

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 β′-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.

Structural basis for transcription inhibition by tagetitoxin

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 Å 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 β and β′ residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg2+ 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 Mg2+ 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.

The carboxy-terminal coiled-coil of the RNA polymerase β′-subunit is the main binding site for Gre factors

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 β′‐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.

Regulation through the RNA Polymerase Secondary Channel STRUCTURAL AND FUNCTIONAL VARIABILITY OF THE COILED-COIL TRANSCRIPTION FACTORS

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 α-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 Å resolution revealing a two-domain architecture closely resembling that of GreA. However, the interdomain orientation is strikingly distinct (∼162° between the two proteins. In contrast to GreA, which has two acidic residues on a well fixed self-stabilized α-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, RNAsynthesis, and pyrophosphorolysis, GreA enhances only the endonucleolytic cleavage.Wepropose 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.

Crystal Structure of a Lysine Biosynthesis Enzyme, LysX, from Thermus thermophilus HB8

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.