Projects:
tmRNA, also known as SsrA, is a small RNA that is present in all bacteria. It has properties of both a tRNA and an mRNA, and it employs this unique structure to regulate gene expression. tmRNA uses an elegant mechanism known as trans-translation to intervene in the translation of substrate proteins and target them for rapid degradation. In this mechanism tmRNA enters the ribosome acting first like a tRNA, and then switches the decoding center of the ribosome from the mRNA to the open reading frame within tmRNA. The result of trans-translation is a protein with a tmRNA-encoded peptide at its C terminus, and this peptide contains multiple proteolytic determinants. We have found that tmRNA regulates initiation of DNA replication by altering the expression of genes encoding replication factors. Our goal is to understand how these replication factors are targeted for trans-translation, and how this activity affects DNA replication.
To study the role of tmRNA in DNA replication, we chose Caulobacter crescentus as model system. In Caulobacter, the G1-S phase transition is coincident with a morphological differentiation from a motile swarmer cell to a stalked cell. Because of this differentiation it is easy to isolate pure populations of cells in G1, and these cells will pass synchronously through the cell cycle. We have previously observed that in Caulobacter lacking tmRNA, the cell cycle is disrupted at the G1-S transition. We identified several DNA replication factors, including the replication initiator protein DnaA, as substrates for tmRNA-mediated trans-translation. To determine the molecular basis for tmRNA activity against DnaA, we will first identify the site of tagging in DnaA, and then make mutations in the corresponding region of the dnaA gene. These mutations will be designed to disrupt predicted sequence elements, remove RNA structural elements, or alter the DnaA protein sequence, and we will analyze the effects of each mutation on tmRNA activity and cellular physiology. In an independent approach, we are using a genetic selection to isolate inhibitors of tmRNA from a library of cyclic peptides. We plan to use these inhibitors to examine the activity of tmRNA as a function of the cell cycle.
Publications:
-
•Cheng L et al. (2007) “Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease.” Protein Sci 16:1535-42.
