Poster abstracts
Poster number 42 submitted by Kara Delbridge
Multi-Color Single-Molecule Tracking of Stop-Codon Readthrough on the Vitamin D Receptor mRNA in Live Cells
Kara Delbridge (Carnegie Mellon University, Department of Biology, Mellon College of Science, Pittsburgh, PA, USA; Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Kelsey E. Bettridge (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Agnes Karasik (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Nicholas R. Guydosh (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA)
Abstract:
Faithful translation of the genetic code by ribosomes requires efficient termination at the end of a coding sequence. However, ribosomes can occasionally insert an amino acid at stop codons and continue translating the 3’ untranslated region (3’ UTR) of the mRNA, leading to the release of a protein with a C-terminal extension. Such readthrough (RT) events can be modulated by the sequence context surrounding the stop codon; several conserved cases with very high RT have been identified and are referred to as programmed RT. One example is the Vitamin D Receptor (VDR), a gene that encodes a master transcription factor that controls the cell’s response to vitamin D and undergoes RT ~7% of the time. To understand the mechanism of programmed RT, we adopted an approach using single-molecule microscopy and a multi-color fluorophore system to observe differences in RT kinetics between mRNAs on a single mRNA. Our system reveals single RT events and that they occurred stochastically about 1% of the time for a normal stop codon, as expected. Surprisingly, we found that programmed RT due to the sequence context from VDR occurred in bursts: one RT event was generally followed by multiple RT events on a given mRNA. This burst process could arise if ribosomes form queues at stop codons and the interactions between ribosomes in the queue change the efficiency of termination. Since ribosome queueing can be altered by stress, such a mechanism could couple stress to programmed RT and regulate the cell’s sensitivity to vitamin D.
Keywords: Microscopy, Ribosome, Readthrough