Quantitative analysis of ribosome-mRNA complexes at different translation stages

Abstract

Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.

Figure 1.

Toeprint analysis of a ribosome–mRNA complex formation. (A) Stable ribosomal (RS) complexes stop reverse transcriptase (RT) at a certain mRNA position generating short cDNA products of specific lengths. Comparison of a (B) classical toeprint experiment—electrophoretic analysis of radioactively labeled products of the primer extension inhibition reaction and (C) alternative approach utilizing fluorescently labeled primers and capillary electrophoresis for detection of the cDNA products. Peaks of fluorescence correspond to the bands on radioautograph.

Figure 2.

Schematic depiction of ribosome–mRNA complexes representing different stages of the translation process. (A) mRNA 5′-end recognition and 5′ UTR scanning. Necessary initiation factors, 40S ribosomal subunit and initiator aminoacyl tRNA are indicated. (B–E) Arrows indicate the factors required for the transition to the consequent stable ribosome–mRNA complex.

Figure 3.

Detection of the initiation ribosome–mRNA 48S complex on natural β-globin mRNA. Lower curves show the case where 40S ribosomal subunits, eIFs: 1, 1A, 2, 3, 4A, 4B, 4F and Met- were present in the reaction mixture. Upper curves are the result of a control reaction where eIF2 was excluded. (A) The original fluorescence readout of the capillary electrophoresis. (B) Same as (A), but the cDNA peaks of fluorescence (blue line) were aligned with the mRNA sequence using Promega CXR size standards (red line). (C) Scaled region of (B) between the 5′-end and toeprint signals, normalized by the integral fluorescence intensity. The aligned β-globin mRNA sequence is shown in the bottom. Three main electrophoregram zones are indicated (see text).

Figure 4.

The dependency of 48S complex yield on the concentration of mRNA in the reconstituted translation system. 40S ribosomal subunits, eIFs: 1, 1A, 2, 3, 4A, 4B, 4F and Met- where present in the reaction mixture. (A) Aligned and normalized electrophoregrams of 48S complex toeprint analysis at indicated β-globin mRNA concentrations. (B) Dependency of the 48S complex concentration (solid line) and relative yield (dashed line) on the β-globin mRNA concentration in the reaction mixture. (C) Comparison of 48S complex formation on different messages. Indicated mRNAs were present in the reaction mixture at the same concentration of 15 nM.

Figure 5.

Specific toeprint patterns of initiation 48S, initiation 80S (elongation-capable), stalled elongation (pre-TC) and termination (TC) ribosomal–mRNA complexes. Numbering of the cDNA peaks corresponds to the position of mRNA nucleotide relative to the triplet located in the P-site of the stalled ribosome.

Figure 6.

Comparison of the toeprint patterns generated by the 48S complexes assembled on different mRNAs (natural β-globin mRNA, non-capped synthetic luciferase-coding mRNAs with A₂₅, Ω TMV and Obe-Luc leaders) at equal reaction conditions. Peaks numbered as in Figure 5.

Figure 7.

Quantitation of translation initiation complexes assembled on β-globin mRNA in non-fractionated RRL (A), and on A₂₅-Luc mRNA in reconstituted eukaryotic initiation system (B). Components of the reaction mixtures used for complex assembly are briefly described on the right. Components required for the assembly of certain complexes are indicated in the bottom of the plots. Only complex-specific zones of the electrophoregrams are shown. Relative distribution of the total complex-specific fluorescence between the peaks is indicated above the plots. Peaks numbered as in Figure 5.

Figure 8.

Quantitation of translation termination reaction on MVHL-STOP mRNA. Plot (A) depicts toeprint pattern of the purified pre-TC, which was used both as a control and as a termination reaction substrate. Only complex-specific zone of the electrophoregram is shown. Plot (B) shows the toeprint pattern after addition of termination factor eRF1 to the pre-TC. Plot (C) is a result of addition of both eRF1 and eRF3.

Figure 9.

Detection of the 48S complex assembly sites in the mixture of natural β-globin mRNA (FAM fluorescence, blue line) and synthetic A₂₅-Luc mRNA (JOE fluorescence, red line). Both fluorescence readouts from FAM and JOE are normalized by the integral fluorescence intensity of the corresponding wavelength. The aligned β-globin and A₂₅-Luc mRNA sequences are shown in the bottom. Complex-specific electrophoregram zones are indicated, and relative yield of the 48S complex assembled on the corresponding mRNA is shown.