Our two main interests are the characterization of the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress and transcriptional regulators whose activities are modified by oxidation and reduction and the identification and characterization of all E. coli noncoding RNAs.

Microbial Responses to Oxidative Stress
Storz, Mukhopadhyay, Outten, Woods, Carmel-Harel, Andrade
One focus of the group is to study how organisms sense environmental signals and transduce the signals into changes in gene expression and cell physiology. Specifically, we are examining the E. coli and S. cerevisiae responses to oxidative stress. Reactive oxygen species can lead to the damage of almost all cell components (DNA, lipid membranes, and proteins) and have been implicated as causative agents in several degenerative diseases. Most organisms have an adaptive response to defend against oxidants. For example, treatment of both bacterial and yeast cells with low doses of hydrogen peroxide results in the induction of a distinct group of proteins, the decreased expression of other proteins, and resistance to killing by subsequent higher doses of hydrogen peroxide.

In bacterial cells, the key regulator of the inducible defenses against hydrogen peroxide is the OxyR transcription factor. We discovered that OxyR is both the sensor and transducer of the oxidative stress signal; the oxidized but not the reduced form of the purified regulator can activate transcription in vitro. OxyR is activated by the formation of an intramolecular disulfide bond between C199 and C208 and is deactivated by enzymatic reduction by glutaredoxin 1 together with glutathione. Structural studies showed that formation of the C199–C208 disulfide bond leads to a large conformational change. Computational and microarray experiments allowed us to identify many of the genes regulated by OxyR.

We now are examining the chemical basis of OxyR sensitivity to hydrogen peroxide and the roles of all of OxyR target genes. Compared with the bacterial response to hydrogen peroxide, less is known about the cellular mechanisms used by higher cells to sense and protect against oxidative damage. To initiate studies of the oxidative stress response in eukaryotes, we constructed isogenic S. cerevisiae strains carrying mutations in known signal transduction pathways and compared the oxidant sensitivities and whole genome expression patterns of these mutants. The studies confirmed that the Yap1 transcription factor is critical for resistance to hydrogen peroxide. We have purified the Yap1 protein and have begun biochemical experiments to characterize this redox-sensitive transcription factor. To identify new components of the eukaryotic response to oxidative stress, we also have been carrying out genetic screens for yeast mutants with altered sensitivities to hydrogen peroxide.

Regulatory RNAs
Storz, Zhang, Kawano
A second focus of the group is to identify untranslated, regulatory RNAs and to elucidate their functions. These noncoding RNAs have been shown to have roles in transcriptional regulation, chromosome replication, RNA processing and modification, mRNA stability and translation, and even protein degradation and translocation. Until recently, however, most noncoding RNAs were discovered fortuitously. Noncoding RNA genes are usually poor targets in genetic screens and have been difficult to detect by direct sequence inspection.

We have been characterizing two previously identified E. coli regulatory RNAs, OxyS and 6S RNA, as well as carrying out screens for additional noncoding RNAs. The OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts as both a global regulator that activates and represses the expression of multiple genes and an antimutator that protects cells against DNA damage. Studies of the fhlA and rpoS targets showed that the OxyS RNA represses translation of these genes. We recently found that OxyS RNA action depends on the Sm-like Hfq protein and that Hfq functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that another abundant noncoding RNA, 6S RNA, binds and modifies RNA polymerase. In addition, we used comparative genomics and microarrays to identify other noncoding RNAs in E. coli. These approaches led to the identification of 17 new regulatory RNAs whose functions we are now characterizing.