Figure 11: Effect of AMP-PNP, a non-hydrolyzable analog of ATP, on mRNA translation in a cell-free system. The wheat germ cell-free system programmed with 5′UTRObelin-GFP-3′UTRTMV mRNA (500 nM) was incubated in batch as described in ‘Materials and Methods’ section at the ATP concentration of 0.1 mM. AMP-PNP was added to the reaction mixture to the final concentration of 2 mM in 10 (filled diamond) or 60 (filled square) minutes after translation start. In control reactions the same amount of ATP was added after 10 min (open square), or the reaction was conducted without any addition (open circle). Arrows indicate the time of AMP-PNP or ATP addition. The synthesis of GFP was recorded by the measurement of fluorescence in 2 µl aliquots taken at the indicated time points.

Kopeina GS, Afonina ZA, Gromova KV, Shirokov VA, Vasiliev VD, Spirin AS - Nucleic Acids Res. (2008)

Bottom Line: The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases.A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate.Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

Affiliation: Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

Abstract: The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.