Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA

Abstract

During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5' untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA.

Keywords: global fitting; polysome formation; ribosome; translation initiation; translational efficiency.

Fig. 1.

Recruitment of the 30S PIC to 5*lpp mRNA. (A) Sequences of the 5′UTR of lpp mRNA and the mutant lpp_AUU mRNA. The start codon of the ORF is shown in red, and an AUG codon at the mRNA 5′ end is in bold. Underlined are the sequences of RNA primers used in the poly(U)–polymerase reaction. (B) Fluorescence change of 5*lpp mRNA upon binding to the 30S PIC. Exp. A: Recruitment of 30S PIC to free 5*lpp mRNA. A schematic of the reaction is shown in the cartoon. AUG is the start codon of the ORF; the rest of the coding region is replaced with a poly(U) sequence; “30S” denotes 30S PIC (0.3 µM) formed of 30S subunits, IF1, IF2–GTP, and IF3. The initial fluorescence of free mRNA is referred to as F₀, and the final fluorescence is referred to as F₁. Exp. B: Recruitment of the 30S PIC to 5*lpp mRNA, which has been translated by the leading 70S ribosome. The 70S IC was formed on 5*lpp (0.05 µM), purified from initiation components and mixed with excess of EF-Tu, EF-G, Phe-tRNA, and GTP to initiate elongation. The 70S EC is stalled at the end of the poly(U) track. The liberated RBS recruits the second 30S PIC (0.3 µM). Exp. C: Recruitment of the 30S PIC to the 70S IC occupying the start codon of 5*lpp mRNA. The 70S IC was prepared as in Exp. B and mixed with the 30S PIC in the absence of the translation mixture. The starting fluorescence F₁ is due to the binding of the first ribosome (Exp. A). The fluorescence of 5*lpp mRNA with two ribosomes bound to the 5′UTR is F₂.

Fig. 2.

The role of proteins S1/S2. Exp. BΔ: Recruitment of the 30S PIC to 5*lpp mRNA, which has been translated by the leading 70SΔ EC. Cartoon represents the schematic of the reaction as in Exp. B in Fig. 1, but performed with the leading ribosome lacking proteins S1 and S2 (shown blue in the cartoon). The 70SΔ IC was formed on 5*lpp mRNA (0.05 µM) mixed with the excess of EF-Tu, EF-G, Phe-tRNA, and GTP to initiate elongation (70SΔ EC). After translation is completed, WT 30S PIC (0.3 µM, white symbol in the cartoon) was added, and the fluorescence change upon recruitment to 5*lpp mRNA was monitored (blue trace; 70SΔ EC-30S). The analogous experiment with the WT 70S EC as a leading ribosome is shown for comparison (black trace). Exp. CΔ: Recruitment of the WT 30S PIC to 5*lpp mRNA when the start codon is occupied by the 70SΔ IC (blue trace) or WT 70S IC (black trace). Cartoon is the same as Exp. C in Fig. 1.

Fig. 3.

The 30S PIC loading during mRNA translation by the leading ribosome. Exp. D: Fluorescence change of 5*lpp mRNA upon translation by the leading 70S EC. The 70S IC (0.05 µM) was rapidly mixed with the translation mix [ternary complex (TC) and EF-G, with GTP]. Cartoon indicates movement of the leading 70S EC upon translation. Exp. E: Same as in Exp. D, but in the presence of the 30S PIC (0.4 µM) bound at the standby site. Cartoon indicates that the 30S PIC movement from the standby site toward the start codon (Fig. 4). Exp. F: Fluorescence of 5*lpp mRNA upon addition of 30S PIC (0.4 µM) and the translation machinery to the purified 70S IC (0.05 µM). Cartoon indicates translation by the leading 70S EC and the movement of the 30S PIC from the stand by site to the vacated start site.

Fig. 4.

Dissecting the initiation kinetics in a polysome. (A) A kinetic mechanism used for global fitting. Errors are SEM of the fit. (B) Deconvoluted time courses for the initial recruitment of the 30S PIC to the standby site (Left); of the rearrangement and accommodation at the start site (Middle); and of the overall reaction (Right). (Top) For 0.05 µM 30S PIC. (Bottom) For 0.4 µM 30S PIC. The experimental time courses used for deconvolution are indicated in each panel. The results of global fit are shown as red lines. Error bars for the translation elongation kinetics are SD (n = 4 independent experiments).

Fig. 5.

Kinetic models of translation initiation. (A) Simplified model of translation initiation on a free mRNA. Initial recruitment is followed by the docking at the standby site. Subsequent accommodation at the start site forms the 30S IC, which after 50S docking can enter the elongation step. (B–D) Loading of the successive ribosomes into a polysome. (B) The second ribosome can bind and dock at the standby site before the leading ribosome moves away from the start codon. Translation is limiting the rate of the second initiation. (C) Queuing of ribosomes at the coding sequence due to, e.g., rare codons preventing loading of the following ribosomes, thereby decreasing the rate of initiation and the overall TE (10, 18, 19). (D) The leading ribosome helps to unwind folded mRNA. If refolding is slower than the start-codon clearance, the following ribosome can bind the mRNA in the polysome faster than the free mRNA (17).