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. Author manuscript; available in PMC: 2015 Feb 21.
Published in final edited form as: Chem Soc Rev. 2013 Aug 22;43(4):1189–1200. doi: 10.1039/c3cs60208g

Figure 10.

Figure 10

a) Design of a 4-color colocalization assay for studying bacterial translation initiation. Cy3-fMet-tRNAfMet (green) and Cy5-labeled IF2 (red) associate with Alexa488™-labeled 30S subunits (blue) bound to mRNA. Subsequently, the Cy3.5-labeled 50S subunit (orange) joins to form a catalytically competent ribosome. b) Representative fluorescence intensity traces indicating the timing of the arrival and departure of each fluorescently labeled component illustrated in part (a) and using the same color scheme. Here, IF2 and fMet-tRNAfMet bind to the 30S-mRNA complex simultaneously to form the 30S PIC. Appearance of the Cy3.5-50S subunit was used to discern correctly assembled PICs from dead end complexes. c) Gathering data from many initiation events revealed that productive PICs can form either by IF2 first, tRNA first, or IF2/tRNA coincident binding pathways. The fractions of PICs formed from each pathway are shown in red. Images and data were reproduced in this figure from reference 39 and with permission from Nature Publishing Group.