Toward a Kinetic Understanding of Eukaryotic Translation

Abstract

The eukaryotic translation pathway has been studied for more than four decades, but the molecular mechanisms that regulate each stage of the pathway are not completely defined. This is in part because we have very little understanding of the kinetic framework for the assembly and disassembly of pathway intermediates. Steps of the pathway are thought to occur in the subsecond to second time frame, but most assays to monitor these events require minutes to hours to complete. Understanding translational control in sufficient detail will therefore require the development of assays that can precisely monitor the kinetics of the translation pathway in real time. Here, we describe the translation pathway from the perspective of its kinetic parameters, discuss advances that are helping us move toward the goal of a rigorous kinetic understanding, and highlight some of the challenges that remain.

Figure 1.

Pathway of eukaryotic translation. The model depicts four main stages of translation. (1) The initiation stage includes the recruitment of the 40S subunit to the m⁷G cap structure, followed by its migration in a 3′ direction. Following start codon selection, the 60S subunit joins to form the 80S ribosome. This stage determines which messenger (mRNA) is selected from the pool and which start codon is chosen to initiate translation. (2) The elongation stage involves decoding the open reading frame (ORF) into a polypeptide chain. This stage can determine the rate at which protein is synthesized when initiation is very rapid (see main text). (3) Translation termination occurs when a termination codon is positioned in the A site of the ribosome, which results in the release of the polypeptide chain. The regulation of termination versus readthrough occurs at this step. (4) Following termination, the 40S and 60S subunits are recycled so that they can take part in another round of translation. For simplicity, the model depicts the recycling of the 40S subunit back onto the same mRNA. The rate of recycling can regulate the availability of ribosomal subunits for translation.

Figure 2.

Cap-dependent translation initiation. The eukaryotic translation initiation pathway is depicted as a series of substeps, many of which are likely to be reversible (see main text for details). Potential sources of regulation for each substep are summarized in boxes and described in more detail in the main text. Selection of messenger RNA (mRNA) by eukaryotic initiation factor (eIF)4F: the mRNA is selected for translation by the binding and accommodation of the eIF4F complex. The accommodation step requires secondary structure in the mRNA to be unwound by the ATP-dependent helicase activity of eIF4A (secondary structure is depicted as “loops” in the 5′UTR). Selection of an mRNA by the 43S PIC: the 43S PIC (40S subunit, eIF1, eIF1A, ternary complex [TC], eIF3, and eIF5) is recruited to the 5′ end of the mRNA through its interaction with eIF4F. Productive recruitment occurs through initial binding and accommodation steps. The accommodation step requires a conformational change in the 40S subunit to a P_OUT/open conformation (described in the main text and depicted as a light gray-colored 40S subunit). Selection of the start codon: the base-pairing between the methionyl-transfer RNA (Met-tRNA_i) and the start codon generates a scanning arrested 43S preinitiation complex (PIC). The arrested complex possesses a P_IN/closed conformation. The final step of the pathway involves the recruitment of the 60S subunit to form the 80S initiation complex (IC). This step is stimulated by the GTPase activity of eIF5B. Following its release from the 40S subunit, the eIF2•GDP complex is recycled to eIF2•GTP by the guanine nucleotide exchange factor (GEF) activity of eIF2B. It should be noted that the possible function of poly(A)-binding protein (PABP) in circularization of the mRNA is not shown for clarity.

Figure 3.

Model of eukaryotic elongation. Elongation is depicted as three main stages: (1) During the decoding step, eEF1A•GTP recruits the appropriate aminoacyl-transfer RNA (aa-tRNA) to the A site of the ribosome. Productive recruitment occurs through initial binding and accommodation steps. The accommodation step requires the hydrolysis of eEF1A-bound GTP, followed by release of eEF1A•GDP. The rate of decoding can be reduced by the presence of rare codons. Following its release, eEF1A•GDP is recycled by the guanine nucleotide exchange factor (GEF) activity of eEF1B. (2) Peptide bond formation between the aa-tRNA in the A site and the peptidyl-tRNA in the P site is catalyzed by the ribosomal RNA (rRNA) through different substeps (not shown). The rate of peptide bond formation is affected by recruitment of eIF5A. (3) The translocation step moves the mRNA relative to the ribosome by one codon. This step requires the recruitment of eEF2•GTP, followed by GTP hydrolysis and release of eEF2•GDP. Potential sources of regulation for each step are summarized in boxes and described in more detail in the main text.

Figure 4.

Translation termination and ribosome recycling. (1) A pre-termination complex is shown to contain a stop codon in the A site of the ribosome. Two possible pathways for subsequent steps are depicted. (2) The recruitment of an eRF1/eRF3•GTP complex to the ribosome in the first step of the termination pathway. (2*) An alternative possibility is that a noncognate aminoacyl transfer RNA (aa-tRNA) is recruited to the A site of the ribosome to continue the elongation stage of translation (stop codon readthrough). Potential sources of regulation for determining which pathway is followed are summarized in the box and described in more detail in the main text. (3) Following the recruitment of eRF1/eRF3•GTP to the ribosome, a number of steps are undertaken to complete translation termination and are described in detail in the main text. Briefly, these steps involve the release of eRF3•GDP, recruitment of ABCE1•ATP, and the release of the polypeptide chain. Finally, the 60S ribosomal subunit is dissociated by the hydrolysis of the ATP bound to ABCE1. As mentioned in the main text, the precise pathway of 40S subunit recycling following termination is poorly understood.