Elizabeth Campbell

Fulminant Jejuno-Ileitis following Ablation of Enteric Glia in Adult Transgenic Mice

To investigate the roles of astroglial cells, we targeted their ablation genetically. Transgenic mice were generated expressing herpes simplex virus thymidine kinase from the mouse glial fibrillary acidic protein (GFAP) promoter. In adult transgenic mice, 2 weeks of subcutaneous treatment with the antiviral agent ganciclovir preferentially ablated transgene-expressing, GFAP-positive glia from the jejunum and ileum, causing a fulminating and fatal jejuno-ileitis. This pathology was independent of bacterial overgrowth and was characterized by increased myeloperoxidase activity, moderate degeneration of myenteric neurons, and intraluminal hemorrhage. These findings demonstrate that enteric glia play an essential role in maintaining the integrity of the bowel and suggest that their loss or dysfunction may contribute to the cellular mechanisms of inflammatory bowel disease.

Crystal Structure of the Bacillus stearothermophilus Anti-σ Factor SpoIIAB with the Sporulation σ Factor σF

Abstract Cell type-specific transcription during Bacillus sporulation is established by σ F . SpoIIAB is an anti-σ that binds and negatively regulates σ F , as well as a serine kinase that phosphorylates and inactivates the anti-anti-σ SpoIIAA. The crystal structure of σ F bound to the SpoIIAB dimer in the low-affinity, ADP form has been determined at 2.9 A resolution. SpoIIAB adopts the GHKL superfamily fold of ATPases and histidine kinases. A domain of σ F contacts both SpoIIAB monomers, while 80% of the σ factor is disordered. The interaction occludes an RNA polymerase binding surface of σ F , explaining the SpoIIAB anti-σ activity. The structure also explains the specificity of SpoIIAB for its target σ factors and, in combination with genetic and biochemical data, provides insight into the mechanism of SpoIIAA anti-anti-σ activity.

Analysis of σ32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli, expression of chaperones and proteases is positively regulated by σ32. Chaperone-mediated negative feedback control of σ32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in σ32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro. We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of σ32.

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β′ subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

Structural biology of bacterial transcription

(RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. At every stage of the transcription cycle, RNAP function is modulated by interactions with extrinsic regulatory factors. Assembling and crystallizing transcription complexes containing extrinsic regulators presents challenges for structural biology. Due to recent advances, cryo-electron microscopy (cryo-EM) now offers a route to structural and mechanistic characterization of these large assemblies. We are using cryo-electron microscopy, in combination with X-ray crystallography and other approaches, to provide a complete characterization of the bacterial transcription cycle.