Nucleic Acids Research Advance Access originally published online on June 26, 2009
Nucleic Acids Research 2009 37(15):5167-5182; doi:10.1093/nar/gkp519
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Nucleic Acids Research, 2009, Vol. 37, No. 15 5167-5182
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing
1Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, 2Department of Physiology and Biophysics, Boston University School of Medicine and 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
*To whom correspondence should be addressed. Tel: +1 718 221 6121; Fax: +1 718 270 2656; Email: tatyana.pestova{at}downstate.edu
Correspondence may also be addressed to Assen Marintchev. Tel: +1 617 638 4295; Fax: +1 617 638 4273; Email: amarint{at}bu.edu
Received May 6, 2009. Revised May 29, 2009. Accepted June 1, 2009.
The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition.