Present address: Fermentas International Inc., Burlington, ON, L7N 3 N4, Canada.
Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3
Article first published online: 22 JAN 2008
© 2008 The Authors
Volume 67, Issue 5, pages 1012–1026, March 2008
How to Cite
Yoo, J.-H. and RajBhandary, U. L. (2008), Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3. Molecular Microbiology, 67: 1012–1026. doi: 10.1111/j.1365-2958.2008.06104.x
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
- Issue published online: 22 JAN 2008
- Article first published online: 22 JAN 2008
- Accepted 21 December, 2007.
Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV–geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA – formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site – are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.