2006 Rustbelt RNA Meeting
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Poster number 33 submitted by Susan Russell

Post-transcriptional modifications in bacterial rRNA and heat shock

Susan Russell (University of Cincinnati), Patrick Limbach (University of Cincinnati)

Abstract:
Post-transcriptional modifications found in ribosomal RNA are thought to be important for both the structure and function of the ribosome. The modified nucleosides of rRNA tend to cluster in the functionally important region of the ribosome and modifications occur in conserved regions. Few rRNA modifying enzymes are known and their regulation is not well understood. At least one modifying enzyme, FtsJ, a specific methyltransferase which creates the 2'-O-methyluridine at U2552 in 23S RNA, has been identified as a heat shock protein.
The heat shock response is a mechanism which allows a cell to cope with a sudden increase in temperature or other environmental stress. The initiation of the heat shock response is transcriptionally regulated by an alternative sigma factor that directs RNA polymerase to different promoter sites which induces a rapid and transient expression of many genes that code for "heat shock proteins". Many of these heat shock proteins are chaperones and proteases, but many have unknown functions.
We are interested in using the sensitivity and specificity of new mass spectrometry-based methods to investigate the effect of the heat shock response on rRNA modification. In this study, Escherichia coli cells were cultured at 30 degrees Celsius to mid log phase and then the temperature was increased to a high but non-lethal temperature (42 degrees Celsius) to induce the heat shock response. Western blotting was used to measure the increase in DnaK, a major heat shock protein, thus verifying the induction of the heat shock response. Isolated rRNA from heat shocked and non-heat shocked cells is digested to nucleosides and the identities and relative amounts of modified nucleosides present in both samples are determined by HPLC and LC-MS approaches.

Keywords: RNA, Heat shock, LC-MS