Global and local depletion of ternary complex limits translational elongation

Abstract

The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms.

Figure 1.

General scheme of translation elongation comprising four discrete processes.

Figure 2.

Global depletion of TC exemplified with the demand of tRNA₂^Gln. (A) The fraction of charged TC decreased by its simultaneous request by m_CAG (0, 100, 1000 ribosomes). The numbers 100 and 1000 indicate that 0.24 and 2.4 times more ribosomes are translating CAG repeats than in the isolated CAG codons which are present normally in the E. coli genome. (B) The increase of the waiting time of the ribosomes for translating a single CAG codon parallels the increase of the concentration of the ribosomes that are involved in the translation of polyGln stretches (m_CAG = 0, 100, 1000).

Figure 3.

During the expression of proteins with polyGln stretches the concentration of the highly requested free tRNA₂^Gln isoacceptor remained unchanged as measured by real-time qRT-PCR. Values are expressed as fold change (log₂; mean ± SD) compared to the control cells expressing only background, chromosomally encoded proteins. tRNA^Trp, tRNA^Met, tRNA₄^Arg and tRNA₂^Ile were used as controls showing also no differences to the control conditions.

Figure 4.

Local depletion of the TC increases the waiting time of a ribosome at a single codon. (A) A radial profile of TC concentration in the vicinity of a ribosome translating CAG repeats at steady state. (B) Waiting time of the ribosomes translating CAG repeats (dashed lines) slightly increased compared to the ribosomes translating isolated CAG codons (solid lines). The simulations were performed assuming that m_CAG [0 (black), 100 (blue) or 1000 (red)] ribosomes are simultaneously translating polyGln stretches in one E. coli cell. (C) Exposure of the cells to osmotic stress (0.6 Osm) severely increased the waiting time of the ribosomes translating isolated CAG codons (solid lines) and CAG repeats (dashed lines).

Figure 5.

The probability of aberrant translation increased by extensive demand of one TC species or global alterations in the TC concentration. (A) Gamma fit of the distribution of the waiting time of ribosomes under optimal growth conditions (black), osmotic stress (red) and overexpressed polyGln protein with m_CAG = 1000 (blue). (B) Large number of ribosomes simultaneously translating polyGln stretches enhances the probability of ribosomal disassembly. (C) Polysomal profile of E. coli cells growing in normal balanced medium (black) and overexpressing Htt exon 1 with 53Gln (blue). To exclude any effect of the plasmid backbone, the control cells were transformed with the empty vector. The peaks corresponding to single ribosomal subunits are designated 30S and 50S, monosomes as M, and peaks representing mRNA occupied by two and more ribosomes are marked as polysomes. (D) Increased molecular crowding by an external osmotic upshift decreases the diffusion coefficient of the TC and subsequently increases the ribosomal drop-off. The macromolecular concentration and its influence on the diffusion properties on the macromolecules was calculated as described (20). Note that in the simulations in (B) and (D), the competition of the release factor for the A-site of the transiently paused ribosomes is considered. (E) Polysomal profile of E. coli cells grown in normal balanced medium (black) and in medium with osmolarity of 0.6 Osm (red). 30S and 50S denote the peaks corresponding to dissociated small and large subunits, respectively.