Abstract
ABSTRACTIn vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation.
Highlights
In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues
Bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond
We have fused the gene coding for the photoswitchable fluorescent protein mEos2 to the C terminus of the endogenous efp gene in E. coli MG1655 and moved the fusion to the VH1000 background for further study (Fig. 1; see Table S1 in the supplemental material) [26]
Summary
In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. During the elongation phase of protein synthesis in prokaryotes, ribosomes are assisted by at least two cofactors that bind and unbind during each translation cycle [1]. Peptides more slowly than other residues [2,3,4] This can lead to translational pausing (ribosome stalling), which in turn can cause premature termination of the elongation phase before a complete protein has been synthesized [2,3,4,5]. Posttranslational -lysylation at residue Lys-34 and an empty ribosomal E site are prerequisite to EF-P binding and activity [1, 4, 13]
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