Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS

ROSAT Evidence for Intrinsic Oxygen Absorption in Cooling Flow Galaxies and Groups

Using spatially resolved, deprojected ROSAT PSPC spectra of 10 of the brightest cooling flow galaxies and groups with low Galactic column densities we have detected intrinsic absorption over energies ~0.4-0.8 keV in half of the sample. Since no intrinsic absorption is indicated for energies below ~0.4 keV, the most reasonable model for the absorber is collisionally ionized gas at temperatures T=10^{5-6} K with most of the absorption arising from ionized states of oxygen but with a significant contribution from carbon and nitrogen. The soft X-ray emission of this warm gas can explain the sub-Galactic column densities of cold gas inferred within the central regions of most of the systems. Attributing the absorption to ionized gas reconciles the large columns of cold H and He inferred from EINSTEIN and ASCA with the lack of such columns inferred from ROSAT. Within the central ~10-20 kpc, where the constraints are most secure, the estimated mass of the ionized absorber is consistent with most (perhaps all) of the matter deposited by a cooling flow over the lifetime of the flow. Since the warm absorber produces no significant H or He absorption the large absorber masses are consistent with the negligible atomic and molecular H inferred from HI and CO observations of cooling flows. It is also found that if T > ~2x10^5 K then the optical and UV emission implied by the warm gas does not violate published constraints. Finally, we discuss how the prediction of warm ionized gas as the product of mass drop-out in these and other cooling flows can be verified with new CHANDRA and XMM observations. (Abridged)Comment: 17 pages (5 figures), Accepted for publication in ApJ, expanded discussion of multiphase spectral models, theoretical implications of warm gas in cooling flows, and the statistical significance of the oxygen absorptio

AGN-Induced Cavities in NGC 1399 and NGC 4649

We present an analysis of archival Chandra and VLA observations of the E0 galaxy NGC 1399 and the E2 galaxy NGC 4649 in which we investigate cavities in the surrounding X-ray emitting medium caused by the central AGN. We calculate the jet power required for the AGN to evacuate these cavities and find values of ~8x10^{41} erg/s and ~14x10^{41} erg/s for the lobes of NGC 1399 and ~7x10^{41} erg/s and ~6x10^{41} erg/s for those of NGC 4649. We also calculate the k/f values for each cavity, where k is the ratio of the total particle energy to that of electrons radiating in the range of 10 MHz to 10 GHz, and f is the volume filling factor of the plasma in the cavity. We find that the values of k/f for the lobes of NGC 1399 are ~93 and ~190, and those of the lobes of NGC 4649 are ~15000 and ~12000. We conclude that the assumed spectrum describes the electron distribution in the lobes of NGC 1399 reasonably well, and that there are few entrained particles. For NGC 4649, either there are many entrained particles or the model spectrum does not accurately describe the population of electrons.Comment: 8 pages, 2 figures, accepted for publication in MNRA