Direct monitoring of initiation factor dynamics through formation of 30S and 70S translation-initiation complexes on a quartz crystal microbalance

Chemistry : a European Journal
Shuntaro TakahashiYoshio Okahata

Abstract

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the aff...Continue Reading

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Related Concepts

AccB protein, E coli
fMet-tRNA(fMet)
Acetyl-CoA Carboxylase
Alkalescens-Dispar Group
Peptide Initiation Factors
Ribosomes
Poly(A) Tail
Transfer RNA
RNA, Transfer, Initiator
Protein Biosynthesis

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