Department of Genetics and Molecular Biology
Centro de Investigacion y de Estudios Avanzados (CINESTAV)
Host: John Hunt
Title: Ribosome stalling during mRNA translation entails accumulation of tRNA fragments in Escherichia coli
Abstract: In search for mRNA fragments protected by ribosomes during translation, we stumbled across fragments of tRNAs. The fragments were present in extracts of wild type Escherichia coli growing under standard conditions and corresponded to 5’ and 3’ tRNA “halves” of nearly all tRNAs in the cell. As we noticed that small amounts of different peptidyl-tRNAs also accumulate under these conditions, we asked whether there was correlation between accumulation of peptidyl-tRNAs and tRNA fragments. In order to answer this question we decided to use minigenes. Minigenes are translatable ORFs containing two to five sense codons whose expression releases peptidyl-tRNA. Under limited peptidyl-tRNA hydrolase activity minigene expression is toxic, presumably because the ribosomes stall at codons cognate to the specific aminoacyl-tRNA accumulated by expression of the particular minigene used. Indeed, increased concentrations of tRNA fragments were observed upon induction of minigene expression, however, the accumulated fragments originated from tRNAs were unrelated to those sequestered by the minigene codons. From these, and other results, we propose that the fragments arise from tRNAs that remain on the stalled ribosomes due to reduced availability of tRNAs cognate to the codon to be translated in the mRNA. I will discuss evidence consistent with this model.