Old dog new tricks, probing enzymatic activity by optimizing a transcription termination assay in RL211 bacteria
Abstract / Artist's Statement
Title: Old dog new tricks, probing enzymatic activity by optimizing a transcription termination assay in RL211 bacteria
ATP-dependent DEAD box RNA helicases are a type of enzyme that unwinds RNA which has adopted a secondary structure. GLH-1 is a DEAD box RNA helicase found in the nematode named C. elegans. GLH-1 is exclusively expressed in germ cells, and is not found in any other part of the worm. Despite a general understanding of GLH-1’s physiological role in proper germline function, GLH-1’s molecular function is currently largely still a mystery. For example, it has been observed that the loss of GLH-1 function can result in catastrophic effects such as temperature sensitive sterility, but it is unclear why (Spike et al. 2008). Beyond GLH-1, there are 3 other GLH-family DEAD box RNA helicases which are named GLH-2, GLH-3, and GLH-4. None of the other 3 members of the GLH helicase family have been tested for enzymatic activity previously.
In order to better understand GLH helicase function, we took a genetic approach. We optimized a transcription termination assay using the bacterial strain RL211. We optimized the temperature of the assay in order to more closely mimic the temperature where GLH enzymes would function in C. elegans. After we optimized this protocol, we tested GLH-1, GLH-2, GLH-3, and GLH-4 in parallel, and report the first enzymatic activity for GLHs 2, 3, and 4 in literature. Our enzymatic activity assay is currently being used to probe further GLH enzymatic activity, and screen for RNA remodeling activity of other RNA-binding proteins in the C. elegans germline.
Old dog new tricks, probing enzymatic activity by optimizing a transcription termination assay in RL211 bacteria
UC South Ballroom
Title: Old dog new tricks, probing enzymatic activity by optimizing a transcription termination assay in RL211 bacteria
ATP-dependent DEAD box RNA helicases are a type of enzyme that unwinds RNA which has adopted a secondary structure. GLH-1 is a DEAD box RNA helicase found in the nematode named C. elegans. GLH-1 is exclusively expressed in germ cells, and is not found in any other part of the worm. Despite a general understanding of GLH-1’s physiological role in proper germline function, GLH-1’s molecular function is currently largely still a mystery. For example, it has been observed that the loss of GLH-1 function can result in catastrophic effects such as temperature sensitive sterility, but it is unclear why (Spike et al. 2008). Beyond GLH-1, there are 3 other GLH-family DEAD box RNA helicases which are named GLH-2, GLH-3, and GLH-4. None of the other 3 members of the GLH helicase family have been tested for enzymatic activity previously.
In order to better understand GLH helicase function, we took a genetic approach. We optimized a transcription termination assay using the bacterial strain RL211. We optimized the temperature of the assay in order to more closely mimic the temperature where GLH enzymes would function in C. elegans. After we optimized this protocol, we tested GLH-1, GLH-2, GLH-3, and GLH-4 in parallel, and report the first enzymatic activity for GLHs 2, 3, and 4 in literature. Our enzymatic activity assay is currently being used to probe further GLH enzymatic activity, and screen for RNA remodeling activity of other RNA-binding proteins in the C. elegans germline.