Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins.

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 (Kd ∼ 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.

The binding site and mechanism of the RNA polymerase inhibitor tagetitoxin: an issue open to debate.

In a recent paper in the Journal of Biological Chemistry, Artsimovitch et al. report a major revision of a crystallographic model and proposed mechanism of the RNA polymerase inhibitor, tagetitoxin. This reassessment is based on theoretical modeling using molecular dynamics simulations. Here, we argue that this theoretical model contradicts experimental results and a published crystal structure cannot exclude several mechanistically distinct alternative models and does not support some major conclusions. We conclude that understanding the tagetitoxin mechanism is beyond the reach of currently available computational simulations and must await input from high-resolution crystal structures of tagetitoxin bound to elongation complex, extensive biochemical studies, or both.

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 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.

The carboxy-terminal coiled-coil of the RNA polymerase beta'-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 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.

Structural basis for converting a general transcription factor into an operon-specific virulence regulator.

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.

Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA.

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.