Abstract
eEF1A, the eukaryotic homologue of bacterial elongation factor Tu, is a well characterized translation elongation factor responsible for delivering aminoacyl-tRNAs to the A-site at the ribosome. Here we show for the first time that eEF1A also associates with the nascent chain distal to the peptidyltransferase center. This is demonstrated for a variety of nascent chains of different lengths and sequences. Interestingly, unlike other ribosome-associated factors, eEF1A also interacts with polypeptides after their release from the ribosome. We demonstrate that eEF1A does not bind to correctly folded full-length proteins but interacts specifically with proteins that are unable to fold correctly in a cytosolic environment. This association was demonstrated both by photo-cross-linking and by a functional refolding assay.
Highlights
Synthesized proteins first encounter the crowded cytosolic environment where total protein concentrations can be as high as 500 mg/ml [1, 2] when they are still nascent chains (NCs)1 being translated on the ribosome
We and others have identified several proteins to be in contact with NCs including signal recognition particle (SRP), DnaJ, nascent polypeptide-associated complex (NAC), and Ssb [17, 19, 20, 30, 31]
Our approach was to combine the co-purification of ribosome-associated factors (RAFs) with a site-specific photo-cross-linking technology where the crosslinker is positioned in a known position of the growing polypeptide chain
Summary
Synthesized proteins first encounter the crowded cytosolic environment where total protein concentrations can be as high as 500 mg/ml [1, 2] when they are still nascent chains (NCs) being translated on the ribosome. We show that eEF1A binds to RNCs and to unfolded polypeptides that are no longer associated with the ribosome but not to correctly folded proteins Based on these data as well as previous reports that eEF1A stimulates ubiquitin-dependent degradation of N-acetylated proteins [23], we suggest that eEF1A may play a role in quality surveillance of newly synthesized proteins
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