Mechanism of cytoplasmic mRNA translation

Abstract

Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings.

Figure 1.

Overview of the steps of translation initiation in the cytoplasm of plants. Once the mRNA has been exported from the nucleus into the cytoplasm it interacts with a cap-binding complex (eIF4F or plant-specific eIFiso4F) at the 5′ end and PABP at the 3′ end. Additional factors, eIF4A and eIF4B are recruited to the mRNA to promote ATP-dependent unwinding of secondary structure prior to interaction with the 43S PIC (pre-initiation complex). The 43S PIC is formed from the 40S ribosome and its associated factors eIF1, eIF1A, eIF3 and eIF5. eIF1, eIF1A, eIF3 and eIF5 form the multi-factor complex (MFC), although it is not clear if this assembles prior to interaction with the ribosome or on the ribosome. Addition of ternary complex (TC) of eIF2·Met-tRNA_i·GTP completes the preparation of the PIC. This then engages the mRNA and its associated factors (eIF4F/eIFiso4F, eIF4A, eIF4B, PABP) to form the 48S scanning complex (open conformation), which functions to scan the 5′ untranslated region of the mRNA in the 5′ to 3′ direction in order to select the AUG (i.e., A/GXXA₊₁UGG). Once the AUG is selected the 48S complex switches to the closed conformation securing the Met-tRNA, in the P-site and ejecting eIF1. eIF5B·GTP binds to the 48S ribosome complex and the process for joining with the 60S subunit commences, and eIFs 6, 5, 3, and 2 exit the 48S ribosome. Completion of joining of the 60S ribosome results in the hydrolysis of eIF5B·GTP and its release along with eIF1A. The now functional 80S ribosome may now start peptide elongation (see Figure 2). The role of eIF2B in guanine nucleotide recycling of eIF2 in plants is unclear at this time (shown with a green line). Note that the factors and ribosomal subunits are not to scale.

Figure 2.

Overview of the steps of plant cytoplasmic translation elongation and termination cycles. The elongating ribosome binds the incoming eEF1A·GTP·aa-tRNA in the A-site. If there is a match between the codon and anticodon of the tRNA, GTP hydrolysis occurs and eEF1A·GDP exits. Peptide bond formation occurs at the peptidyl transferase site; this reaction is mediated by the ribosome. eEF2·GTP binds and hydrolysis of GTP promotes translocation of the mRNA by three nucleotides, moving the now empty tRNA into the E-site, the newly elongated peptide·tRNA into the P-site, generating an empty A-site ready to accept another eEF1A·GTP·aa-tRNA. eEF1A·GDP requires the action of eEF1 B to exchange GDP for GTP. eEF2 does not require a guanine exchange factor to acquire another GTP molecule. It is clear that each step of elongation is expensive in energy. Two GTP are required in the ribosome during elongation and each incoming eEF1A·GTP·aa-tRNA requires the functional equivalent of two ATP molecules to activate the amino acid and add it to the tRNA acceptor arm site. The AMP formed in this process requires two ATP to regenerate back to ATP; thus even though one ATP is consumed in the aminoacylation reaction, two ATP are ultimately consumed. The arrival of the termination codon in the A-site triggers the binding of the eRF1·eRF3·GTP complex into the A-site. Upon GTP hydrolysis, the eRF3·GDP is released. ABCE1 binds at the A-site to the remaining eRF1, promoting release of the polypeptide. Subsequent ATP hydrolysis by ABCE1 dissociates the ribosomal subunits, releasing ABCE1, eRF3, the deacetylated tRNA in the P-site and the mRNA. Note that the factors and ribosomal subunits are not to scale.