Progress 10/01/02 to 09/30/06
Outputs
The aims of this proposal were to identify the physiological function and mechanism(s) of action of the RyeA and RyeB RNAs. Due to several unique features of this pair of RNAs, such knowledge is expected to broaden our global understanding of how RNA molecules can be used in the cell, and how cells respond and survive specific environmental situations. We have uncovered many growth phenotypes associated with the loss of RyeA and RyeB RNAs, many of which are highly suggestive that they function during specific stress responses. Expression profiles for the two RNAs have been determined, and indicate when each RNA is likely to exert its maximum effects. Several genes have been shown to have altered expression in response to loss or overexpression of RyeA and/or RyeB RNAs and further experimentation is being done to indicate which of these genes are direct versus indirect targets for sRNA action. Verification and identification of direct gene targets are anticipated to
highlight the potential mechanisms of action for these sRNAs, which then can be further tested. Already, we are able to place these regulatory molecules into pathways previously unknown to be regulated by small RNAs, expanding the global role of sRNAs in environmental stress responses. Highly conserved elements within these sRNAs have been identified in distantly related bacteria and have lead to parallel studies in other species to continue to expand our knowledge of sRNA regulation which as been most studied in E. coli.
Impacts
As we gain understanding of how cells perceive, respond to, and survive in their natural environments, we will gain insight into novel mechanisms for antimicrobial control. Our work focuses on how two small RNAs assist in cell survival following environmental stress and may highlight important target pathways in this process.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The purpose of this proposal is to identify the function of RyeA and RyeB RNAs which is expected to lead to further understanding of how RNA molecules can be used in the cell, and how cells respond and survive specific environmental situations. Three main objectives were proposed and progress has been made on all fronts. 1. Identification of growth conditions that result in changes to RyeA RNA or RyeB RNA expression. Northern hybridization has determined expression levels of each of these RNAs under various growth conditions including rich or minimal media, exponential or stationary phases of growth, and in response to various environmental stresses. 2. Identification of molecules that interact with RyeA RNA and RyeB RNA. Crosslinking studies suggest RyeB RNA interacts with another RNA species, although further work is required to identify this RNA. Examination of cells deficient for or overexpressing these RNAs have also lead to insights into potential interactions
of these RNAs with other molecules. 3. To elucidate the role of these RNAs in the cell, physiological, genetic and biochemical approaches will be taken dependent on results from the other two objectives. Growth phenotypes of cells lacking ryeA and ryeB genes have been identified and analyzed. The conditions under which these phenotypes are observed strongly indicate that these RNAs are important for survival in response to a number of stresses. The particular observed phenotypes have suggested potential regulatory pathways that these RNAs are involved in. Overexpression or deletion of the individual RNAs, RyeA or RyeB, also has lead to models for interaction between these two RNAs. Future experiments are aimed to provide further elucidation of the details of the mechanisms underlying the function of these two RNAs and their role in cell survival when subjected to multiple environmental stresses.
Impacts
As we gain understanding of how cells perceive, respond to, and survive in their natural environments, we will gain insight into novel mechanisms for antimicrobial control. Our work focuses on how two small RNAs assist in cell survival following environmental stress and may highlight important target pathways in this process.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The purpose of this proposal is to identify the function of RyeA and RyeB RNAs, which is expected to lead to further understanding of how RNA molecules can be used in the cell, and how cells respond and survive in specific environmental situations. Three main objectives were proposed and progress has been made on all fronts. 1. Identification of growth conditions that result in changes to RyeA RNA or RyeB RNA expression. Northern hybridization has determined expression levels of each of these RNAs under various growth conditions including rich or minimal media, exponential or stationary phases of growth, and in response to various environmental stresses. 2. Identification of molecules that interact with RyeA RNA and RyeB RNA. Crosslinking studies suggest RyeB RNA interacts with another RNA species, although further work is required to identify this RNA. Examination of cells deficient for or overexpressing these RNAs has also lead to insights into potential
interactions of these RNAs with other molecules. 3. To elucidate the role of these RNAs in the cell, physiological, genetic and biochemical approaches will be taken dependent on results from the other two objectives. Growth phenotypes of cells lacking ryeA and ryeB genes have been analyzed and have suggested specific conditions of growth when these RNAs are important. These data are supported by use of plasmids expressing each RNA independently. Future experiments are aimed to provide further elucidation of the details of how these two RNAs function in response to environmental stress.
Impacts
As we gain understanding of how cells perceive, respond to, and survive in their natural environments, we will gain insight into novel mechanisms for antimicrobial control. Our work focuses on how two small RNAs assist in cell survival following environmental stress and may highlight important target pathways in this process.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The purpose of this proposal is to identify the function of RyeA and RyeB RNAs which is expected to lead to further understanding of how RNA molecules can be used in the cell, and how cells respond and survive specific environmental situations. Three main objectives were proposed and progress has been made on all fronts. 1. Identification of growth conditions that result in changes to RyeA RNA or RyeB RNA expression. Northern hybridization has determined expression levels of each of these RNAs under various growth conditions including rich or minimal media, exponential or stationary phases of growth, and in response to various environmental stresses. 2. Identification of molecules that interact with RyeA RNA and RyeB RNA. Crosslinking studies suggest RyeB RNA interacts with another RNA species, although further work is required to identify this RNA. Future biochemical fractionation techniques such as density gradients, anti-sense selection of each RNA with bound
factors, and immunoprecipitation with identified binding-proteins will be used. Protein candidates will be identified using mass spectrometry, and nucleic acid candidates will be identified by direct sequencing. 3. To elucidate the role of these RNAs in the cell, physiological, genetic and biochemical approaches will be taken dependent on results from the other two objectives. Cells lacking ryeA and ryeB genes have been constructed to examine phenotypes associated with their loss of function. Likewise, plasmids expressing each RNA have been generated to allow control of RNA levels. Preliminary growth studies suggest one or both of these RNAs are critical in stress response. Future experiments are aimed at elucidating the details of the roles of these two RNAs in these responses.
Impacts
As we gain understanding of how cells perceive, respond to, and survive in their natural environments, we will gain insight into novel mechanisms for antimicrobial control. Our work focuses on how two small RNAs assist in cell survival following environmental stress and may highlight important target pathways in this process.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
This project is still in the very preliminary stages as it began on 10/01/2002. We have initiated experiments outlined in Specific Aim 1 to examine when during a normal growth curve RyeA and RyeB RNAs are expressed, as well as during what types of environmental stresses. These experiments are designed to highlight important time frames when these RNAs are functioning. We also are currently developing bacterial strains in which RyeA and RyeB RNAs are overexpressed or non-functional to be used as tools for further characterization of these two small RNAs as outlined in Specific Aims 2 and 3.
Impacts
Small RNA molecules are key regulators that allow more efficient microbial survival under stressful conditions such as those encountered in the natural environment. We are characterizing two new small RNA genes, ryeA and ryeB, to elucidate when and how they function in bacterial growth and survival. Further understanding of how bacteria are able to survive harsh environments will undoubtedly lead to improvements in our ability to control unwanted microbial growth critical to the efficient survival of many agriculturally important crops and animals.
Publications
- No publications reported this period
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