The function, structure and regulation of E. coli peptide chain release factors

The termination of protein synthesis in Escherichia coli depends upon the soluble protein factors RF1 or RF2. RF1 catalyzes UAG and UAA dependent termination, while RF2 catalyzes UGA and UAA dependent termination. The proteins have been purified to homogeneity, their respective genes isolated, and their primary structures deduced from the DNA sequences. The sequences reveal considerable conserved homology, presumably reflecting functional similarities and a common ancestral origin. The RFs are encoded as single copy genes on the bacterial chromosome. RF2 exhibits autogenous regulation in an in vitro translation system. The mechanism of autoregulation appears to be an in-frame UGA stop codon that requires a 1+ frameshift for the continued synthesis of the protein. Frameshifting prior to the inframe stop codon occurs at a remarkably high frequency by an unknown mechanism. Future studies will be directed at understanding how RFs interact with the ribosomal components, and further defining the mechanism of RF2 frameshifting.     

Does protein synthesis occur in the nucleus?

Although it is universally accepted that protein synthesis occurs in the cytoplasm, the possibility that translation can also take place in the nucleus has been hotly debated. Reports have been published claiming to demonstrate nuclear translation, but alternative explanations for these results have not been excluded, and other experiments argue against it. Much of the appeal of nuclear translation is that functional proofreading of newly made mRNAs in the nucleus would provide an efficient way to monitor mRNAs for the presence of premature termination codons, thereby avoiding the synthesis of deleterious proteins. mRNAs that are still in the nucleus-associated fraction of cells are subject to translational proofreading resulting in nonsense-mediated mRNA decay and perhaps nonsense-associated alternate splicing. However, these mRNAs are likely to be in the perinuclear cytoplasm rather than within the nucleus. Therefore, in the absence of additional evidence, we conclude that nuclear translation is unlikely to occur.     

mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation

Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.     

A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis

Low temperatures lead to the inhibition of sucrose synthesis and photosynthesis. The biochemical and physiological adaptations of plants to low temperatures include the post-translational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in the leaf protein content. Recent progress has been made in understanding both the signals that trigger these processes and how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation.     

Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen

The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.     

A mathematical modelling framework for elucidating the role of feedback control in translation termination

Translation is the final stage of gene expression where messenger RNA is used as a template for protein polymerization from appropriate amino acids. Release of the completed protein requires a release factor protein acting at the termination/stop codon to liberate it. In this paper we focus on a complex feedback control mechanism involved in the translation and synthesis of release factor proteins, which has been observed in different systems. These release factor proteins are involved in the termination stage of their own translation. Further, mutations in the release factor gene can result in a premature stop codon. In this case translation can result either in early termination and the production of a truncated protein or readthrough of the premature stop codon and production of the complete release factor protein. Thus during translation of the release factor mRNA containing a premature stop codon, the full length protein negatively regulates its production by its action on a premature stop codon, while positively regulating its production by its action on the regular stop codon. This paper develops a mathematical modelling framework to investigate this complex feedback control system involved in translation. A series of models is established to carefully investigate the role of individual mechanisms and how they work together. The steady state and dynamic behaviour of the resulting models are examined both analytically and numerically.     

Translational termination comes of age

Translational termination has been a largely ignored aspect of protein synthesis for many years. However, the recent identification of new release-factor genes, the mapping of release-factor functional sites and in vitro reconstitution experiments have provided a deeper understanding of the termination mechanism. In addition, protein-protein interactions among release factors and with other proteins have been revealed. The three-dimensional structures of a prokaryotic ribosome recycling factor and eukaryotic release factor 1 (eRF1) mimic the shape of transfer RNA, indicating that they bind to the same ribosomal site. Post-termination events in bacteria have been clarified, linking termination, ribosomal recycling and translation initiation.     

RNA-protein interactions at the initial and terminal stages of protein biosynthesis as investigated by Lev Kisselev (On the occasion of his 70th anniversary)

This review highlights studies by Lev L. Kisselev and his colleagues on the initial and terminal stages of protein biosynthesis, which cover the period of the last 45 years (1961-2006). They investigated spatial structure of tRNAs, structure and functions of aminoacyl-tRNA-synthetases of higher organisms, and the final step of protein synthesis, termination of translation. L. Kisselev and his team have made three major contributions to these fields of molecular biology; (i) they proposed the hypothesis on the role of anticodon triplet of tRNA in recognition by cognate aminoacyl-tRNA synthetase, which has been experimentally confirmed and is now included in textbooks; (ii) identified primary structures and functions of two eukaryotic protein factors (eRF1 and eRF3) playing a pivotal role in translation termination; (iii) characterized a structural basis for stop codon recognition by eRF1 within the ribosome and discovered the negative structural elements of eRF1, limiting its recognition of one or two stop-codons.     

The elongation, termination, and recycling phases of translation in eukaryotes

This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.     

Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency

Translation termination is the final step that completes the synthesis of a polypeptide. Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein. We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upfl-like family of helicases that is involved in modulating translation termination. MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination. Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain. Nonsense suppression is apparently not due to induction of [PSI+], even though cooverexpression of HSP104 alleviated the nonsense suppression phenotype observed in cells overexpressing MTT1, suggesting a more direct role of Hsp104p in the translation termination process. The MTT1 gene product was shown to interact with translation termination factors and is localized to polysomes. Taken together, these results indicate that at least two members of a family of RNA helicases modulate translation termination efficiency in cells.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.