We investigate how cells protect the integrity of their macromolecules: DNA, RNA, and proteins. Every cell, from free-living bacteria to neurons, constantly experiences intrinsic and external threats to the integrity of its genome and proteome. For example, DNA can be damaged by intrinsic conflicts between transcription and DNA replication or repair assemblies that collide at numerous places along the chromosome. Proteins are prone to spontaneous misfolding and aggregation unless shielded by molecular chaperones. Environmental assaults in the form of chemical (e.g., oxidants and toxins) or physical (e.g., heat) stress further contribute to cellular damage. Understanding the fundamental mechanisms by which cells resolve inherent molecular conflicts and protect their constituents has implications for numerous pathological conditions and aging. The field of cellular stress response is very broad. We are concentrating our efforts on four major areas.
We investigate the mechanisms by which the transcription apparatus responds to environmental challenges such as starvation, oxidative stress, DNA damage, etc., in diverse bacterial systems. We are particularly interested in the elaborate mechanics of the elongation phase of the transcription cycle.
Our lab has developed various biochemical and protein chemical tools to examine how RNA polymerase (RNAP) moves along its DNA template, how it responds to regulatory factors and signals encoded in RNA and DNA, and how it terminates transcription.
Many of our investigations evolved from our initial finding of RNAP backtracking: the reverse sliding of the enzyme along DNA and RNA at numerous sites along a chromosome (Figure 1). Backtracking plays the central role in controlling the rate of transcription elongation and the processes coupled to it, e.g., cotranscriptional RNA folding, processing, and termination. The majority of the regulatory pauses of RNAP that control gene expression in bacteria and eukaryotes are backtracking events.
Backtracking also provides a mechanistic link between transcription elongation and DNA replication and repair and mRNA translation. Traditionally, these activities have been studied separately, but they occur simultaneously in time and space in the cell and influence one another dramatically. For example, we showed that in bacteria the moving ribosome controls the speed of RNAP by "pushing" it forward (i.e., preventing backtracking; Figure 1). Such cooperation between RNAP and the ribosome explains how bacteria can precisely adjust transcriptional yield to transcriptional needs under various growth and stress conditions. It also contributes to genomic stability by preventing deleterious collisions between backtracked RNAP and the replisome (Figure 1).
More recently, we showed that RNAP backtracking is also a crucial part of the DNA repair mechanism. When DNA is damaged, e.g., by ultraviolet light, a specialized helicase (UvrD) binds and pulls RNAP backward from the lesion site, thereby exposing the lesion to the repair machinery.
These examples emphasize the importance of interaction between the molecular machines involved in transcription, RNA processing, DNA replication and repair, and mRNA translation. Our proteomic data suggest that RNAP provides interfaces for interactions with many factors involved in these processes; we have only begun to understand the structure and function of these interactions.
Nitric oxide (NO) and hydrogen sulfide (H2S) are well-established mammalian signaling molecules. However, their functions in common bacteria are largely unknown. We showed that various bacterial species generate NO and H2S enzymatically from arginine and cysteine, respectively, and that these two gases function to protect bacteria from oxidative stress and a wide range of antibiotics (Figure 2).
Moreover, bacterial NO and H2S act together to protect pathogens such as Staphylococcus aureus and Bacillus anthracis from immune attack. Microarray analyses indicated that endogenous NO and H2S regulate hundreds of bacterial genes. We are elucidating the molecular mechanisms of such regulation at the transcriptional and post-transcriptional levels.
We also study how these gases are regulated in bacteria and how they affect key physiological and clinically relevant processes, such as biofilm formation, swarming, motility, sporulation, and quorum sensing.
Bacteria-Host Interaction and Aging
We use the round worm Caenorhabditis elegans as a model system to investigate how commensal bacteria affect the physiology and aging of their hosts.
Bacteria are not merely food for C. elegans, they also colonize adult worms and can dramatically influence the animal's behavior and life span. Similarly, billions of bacteria colonize the mammalian digestive tract, including that of humans. However, their effects on human well-being and aging remain largely unknown.
In the laboratory, C. elegans are fed almost exclusively on Escherichia coli. However, in their natural habitat these nematodes consume soil bacteria, such as bacilli. Remarkably, worms fed Bacillus subtilis live much longer than those fed E. coli. We identified metabolites produced by B. subtilis that extend the life span of the worm and increase the worm's resistance to environmental stressors such as heat and oxidants. One of these metabolites is NO. We showed that bacteria-derived NO, generated inside the worm, diffuses into the animal's tissues and activates a defined set of cytoprotective genes, rendering C. elegans more resistant to stress and allowing it to live longer (Figure 3).
We are designing probiotic strains of bacteria that significantly extend the nematode life span. We are also investigating the mechanisms by which bacteria-derived small molecules increase nematode longevity. Separately, we study how different forms of commensal bacterial communities (e.g., biofilms) influence the nematode life span and resistance to stress and infection.
Heat Shock Response
Heat shock genes encode molecular chaperones and other cytoprotective molecules that prevent accumulation of damaged proteins. In eukaryote
We identified a nucleoprotein complex that contains the translation elongation factor eEF1A1 and a large noncoding RNA (HSR1) that is required for HSF1 activation. We proposed that HSR1 acts as a molecular thermosensor that determines the temperature threshold for the heat shock response (HSR). We also showed that eEF1A1 orchestrates the entire process of the HSR, from the transcriptional activation of heat shock genes to the stabilization, transport, and translation of their mRNAs (Figure 4). We continue to investigate the complex mechanism of HSR at the transcriptional and post-transcriptional levels.
The results of these studies have implications for numerous physiological conditions associated with protein misfolding, including various types
Grants from the National Institutes of Health, Robertson Foundation, and Blavatnik Family Foundation provided partial support for some of these projects
As of February 25, 2016