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Heterogeneous pathways and timing of factor departure during translation initiation.

Tsai A, Petrov A, Marshall RA, Korlach J, Uemura S, Puglisi JD - Nature (2012)

Bottom Line: Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition.IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex.These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.

ABSTRACT
The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

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50S subunit joining to 30S PICa. The appearance of a stable Cy5 signal is used to identify the arrival of the 50S subunit (Methods 5). The time until 50S arrival and the length of the 50S signal are then characterized.b. The arrival times of a 50S subunit to and the observed 50S subunit lifetimes on a 30S PIC are fitted to single-exponential functions and plotted with s.d. error bars. The presence of IF2 is critical for efficiently and stably forming 70S complexes. From left to right for both panels, n = 246, n = 283, n = 451, n = 262, and n = 253.
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Figure 2: 50S subunit joining to 30S PICa. The appearance of a stable Cy5 signal is used to identify the arrival of the 50S subunit (Methods 5). The time until 50S arrival and the length of the 50S signal are then characterized.b. The arrival times of a 50S subunit to and the observed 50S subunit lifetimes on a 30S PIC are fitted to single-exponential functions and plotted with s.d. error bars. The presence of IF2 is critical for efficiently and stably forming 70S complexes. From left to right for both panels, n = 246, n = 283, n = 451, n = 262, and n = 253.

Mentions: 50S subunit joining to a 30S PIC to form a 70S initiation complex (70S IC) is the second major molecular event of initiation. To track subunit joining, we used 50S subunits labeled with single dyes, which were shown to be functional in prior intersubunit FRET studies11,12. We delivered Cy5-50S subunits to immobilized 30S PICs (Methods 5). IF2 in the presence of GTP drives rapid, stable subunit joining8 (Figure 2a). At 2 μM IF2(GTP), Cy5-50S subunits joined rapidly to 30S PICs with an observed kon = 1×106 M−1s−1 (τ = 9 s), forming complexes whose lifetime was limited by photobleaching (τ = 38 s) (Figure 2b, Supplementary Figure 4 & Supplementary Text 2). In accordance with previous studies8, omitting IF2 resulted in slow and unstable subunit joining, decreasing kon to 0.3×106 M−1s−1 (τ = 29 s) and 50S lifetime to τ = 6 s. In the presence of IF2 and non-hydrolysable GDPNP, 50S subunit arrival rate was similar to that of IF2(GTP). However, 50S subunit stability decreased to a lifetime of τ = 28 s, consistent with prior intersubunit FRET results that GDPNP-bound IF2 can guide stable subunit joining without GTP hydrolysis11,13. Addition of the other two initiation factors, IF1 and IF3, at 1μM each to 2 μM IF2(GTP) did not appreciably change the kon or the lifetime of the 50S subunit on our model mRNA.


Heterogeneous pathways and timing of factor departure during translation initiation.

Tsai A, Petrov A, Marshall RA, Korlach J, Uemura S, Puglisi JD - Nature (2012)

50S subunit joining to 30S PICa. The appearance of a stable Cy5 signal is used to identify the arrival of the 50S subunit (Methods 5). The time until 50S arrival and the length of the 50S signal are then characterized.b. The arrival times of a 50S subunit to and the observed 50S subunit lifetimes on a 30S PIC are fitted to single-exponential functions and plotted with s.d. error bars. The presence of IF2 is critical for efficiently and stably forming 70S complexes. From left to right for both panels, n = 246, n = 283, n = 451, n = 262, and n = 253.
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Related In: Results  -  Collection

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Figure 2: 50S subunit joining to 30S PICa. The appearance of a stable Cy5 signal is used to identify the arrival of the 50S subunit (Methods 5). The time until 50S arrival and the length of the 50S signal are then characterized.b. The arrival times of a 50S subunit to and the observed 50S subunit lifetimes on a 30S PIC are fitted to single-exponential functions and plotted with s.d. error bars. The presence of IF2 is critical for efficiently and stably forming 70S complexes. From left to right for both panels, n = 246, n = 283, n = 451, n = 262, and n = 253.
Mentions: 50S subunit joining to a 30S PIC to form a 70S initiation complex (70S IC) is the second major molecular event of initiation. To track subunit joining, we used 50S subunits labeled with single dyes, which were shown to be functional in prior intersubunit FRET studies11,12. We delivered Cy5-50S subunits to immobilized 30S PICs (Methods 5). IF2 in the presence of GTP drives rapid, stable subunit joining8 (Figure 2a). At 2 μM IF2(GTP), Cy5-50S subunits joined rapidly to 30S PICs with an observed kon = 1×106 M−1s−1 (τ = 9 s), forming complexes whose lifetime was limited by photobleaching (τ = 38 s) (Figure 2b, Supplementary Figure 4 & Supplementary Text 2). In accordance with previous studies8, omitting IF2 resulted in slow and unstable subunit joining, decreasing kon to 0.3×106 M−1s−1 (τ = 29 s) and 50S lifetime to τ = 6 s. In the presence of IF2 and non-hydrolysable GDPNP, 50S subunit arrival rate was similar to that of IF2(GTP). However, 50S subunit stability decreased to a lifetime of τ = 28 s, consistent with prior intersubunit FRET results that GDPNP-bound IF2 can guide stable subunit joining without GTP hydrolysis11,13. Addition of the other two initiation factors, IF1 and IF3, at 1μM each to 2 μM IF2(GTP) did not appreciably change the kon or the lifetime of the 50S subunit on our model mRNA.

Bottom Line: Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition.IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex.These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.

ABSTRACT
The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

Show MeSH
Related in: MedlinePlus