Faculty Bibliography

Most bacteria generate nitric oxide (NO) either aerobically by NO synthases or anaerobically from nitrite. Far from being a mere by-product of nitrate respiration, bacterial NO has diverse physiological roles. Many proteins undergo NO-mediated posttranslational modification (S-nitrosylation) in anaerobically grown Escherichia coli. The regulation of one such protein, OxyR, represents a redox signaling paradigm in which the same transcription factor controls different protective genes depending on its S-nitrosylation versus S-oxidation status. We discuss a structural model that may explain the remarkable stability and specificity of OxyR S-nitrosylation.

Riboswitches are RNA sensors that regulate gene expression upon binding specific metabolites or ions. Bacterial riboswitches control gene expression primarily by promoting intrinsic transcription termination or by inhibiting translation initiation. We now report a third general mechanism of riboswitch action: governing the ability of the RNA-dependent helicase Rho to terminate transcription. We establish that Rho promotes transcription termination in the Mg(2+)-sensing mgtA riboswitch from Salmonella enterica serovar Typhimurium and the flavin mononucleotide-sensing ribB riboswitch from Escherichia coli when the corresponding riboswitch ligands are present. The Rho-specific inhibitor bicyclomycin enabled transcription of the coding regions at these two loci in bacteria experiencing repressing concentrations of the riboswitch ligands in vivo. A mutation in the mgtA leader that favors the "high Mg(2+)" conformation of the riboswitch promoted Rho-dependent transcription termination in vivo and in vitro and enhanced the ability of the RNA to stimulate Rho's ATPase activity in vitro. These effects were overcome by mutations in a C-rich region of the mRNA that is alternately folded at high and low Mg(2+), suggesting a role for this region in regulating the activity of Rho. Our results reveal a potentially widespread mode of gene regulation whereby riboswitches dictate whether a protein effector can interact with the transcription machinery to prematurely terminate transcription.

In their commentary, Klyuyev and Vassylyev dispute a model of transcription inhibition by tagetitoxin (Tgt) proposed by us based on biochemical analysis and computational docking. We maintain that, although an alternative explanation can be provided for any single observation reported by us, taken together our results support a model in which Tgt acts by trapping the trigger loop (TL) in an inactive state (Artsimovitch et al.). ( 1) This model is consistent with all the data collected with a physiological target for the inhibitor, the transcription elongation complex (EC). The Tgt-binding pose in our model is indeed different from that observed in the structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in the absence of nucleic acids (Vassylyev et al. Nat Struct Mol Biol 2005; 12:1086). The latter can hardly be considered a dogma because (1) RNAP undergoes conformational changes in the course of the transcription cycle and during catalysis and (2) small molecules containing phosphates likely bind to several sites on RNAP, with the crystallographic site/pose not necessarily being the one most relevant mechanistically. Furthermore, the model proposed based on the Tgt/holoenzyme structure does not explain the inhibitor's effects on transcript elongation and RNAP translocation. These arguments necessitate further inquiry into the mechanism of inhibition by Tgt by techniques orthogonal to X-ray crystallography. In our opinion, elucidation of a molecular mechanism of any RNAP inhibitor and the follow-up design of more potent derivatives requires a combination of approaches, including genetics, biochemistry, biophysics, X-ray crystallography and computational analysis.

Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine beta-synthase, cystathionine gamma-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics

Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the beta' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a "slow" enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics.

Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional collisions with backtracked (arrested) ECs lead to DNA double-strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to avoid replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance

The transcription of the genetic information encoded in DNA into RNA is performed by RNA polymerase (RNAP), a complex molecular motor, highly conserved across species. Despite remarkable progress in single-molecule techniques revealing important mechanistic details of transcription elongation (TE) with up to base-pair resolution, some of the results and interpretations of these studies are difficult to reconcile, and have not yet led to a minimal unified picture of transcription. We propose a simple model that accounts quantitatively for many of the experimental observations. This model belongs to the class of isothermal ratchet models of TE involving the thermally driven stochastic backward and forward motion (backtracking and forward tracking) of RNAP along DNA between single-nucleotide incorporation events. We uncover two essential features for the success of the model. The first is an intermediate state separating the productive elongation pathway from nonelongating backtracked states. The rates of entering and exiting this intermediate state modulate pausing by RNAP. The second crucial ingredient of the model is the cotranscriptional folding of the RNA transcript, sterically inhibiting the extent of backtracking. This model resolves several apparent differences between single-molecule studies and provides a framework for future work on TE

Brain deterioration resulting from protein folding diseases, such as the Alzheimer's disease (AD), is one of the leading causes of morbidity and mortality in the aging human population. Heat shock proteins (HSPs) constitute the major cellular quality control system for proteins that mitigates the pathological burden of neurotoxic protein fibrils and aggregates. However, the therapeutic effect of HSPs has not been tested in a relevant setting. Here we report the dramatic neuroprotective effect of recombinant human Hsp70 in the bilateral olfactory bulbectomy (OBE) mouse model. We show that intranasally administered Hsp70 rapidly enters the afflicted brain regions and mitigates multiple AD-like morphological and cognitive abnormalities observed in OBE animals. In particular, it normalized the density of neurons in the hippocampus, which correlated with the diminished accumulation of amyloid b (Ab) peptide. Consistently, Hsp70 also fully protected spatial memory, which remained at the level of control animals for at least eight months following treatment. The long-lasting therapeutic effect of Hsp70 suggests a novel mechanism of action and establishes it as a practical and potent agent for treatment of neurodegenerative diseases associated with abnormal protein biogenesis and cognitive disturbances, such as AD, for which neuroprotective therapy is urgently needed

Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5'/3'-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes