Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 3' side

The downstream region (DR) located immediately after the initiation codon acts as a translational enhancer and depending on its sequence gene expression can vary considerably. In order to determine the influence of the DR on the apparent translation initiation, we have analyzed several naturally occurring DRs (a stretch of five codons) in a lacZ reporter gene. The efficiency of expression, associated with these DRs did not show any correlation to the expression levels connected with the natural genes. Changes of the iso-codon composition in the DR, thus maintaining the amino acid sequence in the gene product, gave significant variations in gene expression. Thus, the messenger RNA base sequence, and not the encoded amino acid sequence, in the early coding region is the determinant for the apparent efficiency of translation initiation and/or early elongation.     

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.     

RNA chaperone activity of translation initiation factor IF1

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.     

Characterization of an Initiating Cell-Free Protein Synthesis System Derived from Rabbit Brain

Abstract: Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro : (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [³⁵S]Met-tRNA_f to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions for amino acid incorporation in this system were 4 mM-Mg²⁺, 140 mM-K⁺, and pH 7.55. Incorporation was dependent on the addition of ATP, GTP, and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.     

The +4G site in Kozak consensus is not related to the efficiency of translation initiation

The optimal context for translation initiation in mammalian species is GCCRCCaugG (where R = purine and "aug" is the initiation codon), with the -3R and +4G being particularly important. The presence of +4G has been interpreted as necessary for efficient translation initiation. Accumulated experimental and bioinformatic evidence has suggested an alternative explanation based on amino acid constraint on the second codon, i.e., amino acid Ala or Gly are needed as the second amino acid in the nascent peptide for the cleavage of the initiator Met, and the consequent overuse of Ala and Gly codons (GCN and GGN) leads to the +4G consensus. I performed a critical test of these alternative hypotheses on +4G based on 34169 human protein-coding genes and published gene expression data. The result shows that the prevalence of +4G is not related to translation initiation. Among the five G-starting codons, only alanine codons (GCN), and glycine codons (GGN) to a much smaller extent, are overrepresented at the second codon, whereas the other three codons are not overrepresented. While highly expressed genes have more +4G than lowly expressed genes, the difference is caused by GCN and GGN codons at the second codon. These results are inconsistent with +4G being needed for efficient translation initiation, but consistent with the proposal of amino acid constraint hypothesis.     

Physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors IF1 and IF2

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.     

Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system

Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.     

Exogenous amino acids regulate trophectoderm differentiation in the mouse blastocyst through an mTOR-dependent pathway.

At the late blastocyst stage, the epithelial trophectoderm cells of the mammalian embryo undergo a phenotypic change that allows them to invade into the uterine stroma and make contact with the maternal circulation. This step can be regulated in vitro by the availability of amino acids. Embryos cultured in defined medium lacking amino acids cannot form trophoblast cell outgrowths on fibronectin, an in vitro model of implantation, but remain viable for up to 3 days in culture and will form outgrowths when transferred into complete medium. The amino acid requirement is a developmentally regulated permissive event that occurs during a 4- to 8-h period at the early blastocyst stage. Amino acids affect spreading competence specifically by regulating the onset of protrusive activity and not the onset of integrin activation. Rapamycin, a specific inhibitor of the kinase mTOR/FRAP/RAFT1, blocks amino acid stimulation of embryo outgrowth, demonstrating that mTOR is required for the initiation of trophectoderm protrusive activity. Inhibition of global protein translation with cycloheximide also inhibits amino acid-dependent signals, suggesting that mTOR regulates the translation of proteins required for trophoblast differentiation. Our data suggest that mTOR activity has a developmental regulatory function in trophectoderm differentiation that may serve to coordinate embryo and uterus at the time of implantation.     

Both the 5'' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro.

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediating translation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence was replaced with either optimized or compromised leader sequences, and translational efficiency was measured for six different coding regions. Translation was most efficient with a leader containing the 5' untranslated region from Xenopus beta-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybrid was observed to increase translation of the various coding regions as much as 300-fold. The translational efficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions that increased leader efficiency results from higher affinity of the leader for a limiting factor, our experiments suggest that increased translation from the beta-globin hybrid leader sequence results from more rapid initiation of translation.     

A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.     

Invited Review: Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise.

Protein synthesis in skeletal muscle is modulated in response to a variety of stimuli. Two stimuli receiving a great deal of recent attention are increased amino acid availability and exercise. Both of these effectors stimulate protein synthesis in part through activation of translation initiation. However, the full response of translation initiation and protein synthesis to either effector is not observed in the absence of a minimal concentration of insulin. The combination of insulin and either increased amino acid availability or endurance exercise stimulates translation initiation and protein synthesis in part through activation of the ribosomal protein S6 protein kinase S6K1 as well as through enhanced association of eukaryotic initiation factor eIF4G with eIF4E, an event that promotes binding of mRNA to the ribosome. In contrast, insulin in combination with resistance exercise stimulates translation initiation and protein synthesis through enhanced activity of a guanine nucleotide exchange protein referred to as eIF2B. In both cases, the amount of insulin required for the effects is low, and a concentration of the hormone that approximates that observed in fasting animals is sufficient for maximal stimulation. This review summarizes the results of a number of recent studies that have helped to establish our present understanding of the interactions of insulin, amino acids, and exercise in the regulation of protein synthesis in skeletal muscle.     

Effect of ethionine on the in vitro synthesis and degradation of mitochondrial translation products in yeast

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.     

Reconstitution of eukaryotic translation elongation in vitro following initiation by internal ribosomal entry

The ability to reconstitute different stages of eukaryotic translation process in vitro is a prerequisite for detailed biochemical analysis of their mechanisms. Reconstitution of elongation and subsequent processes such as termination and recycling on natural mRNAs translated by the cap-dependent mechanism is very complicated, and has not so far been done because of the necessity to first reconstitute the process of translation initiation, which is the most complex stage of eukaryotic translation, which requires at least nine initiation factors. The recent discovery of internal ribosomal entry sites (IRESs) in the intergenic region (IGR) of the genomes of dicistroviruses such as cricket paralysis virus (CrPV) and Plautia stali intestine virus (PSIV) that mediate initiation of translation by a mechanism that does not involve aminoacylated initiator tRNA (Met-tRNA(i)Met) or any initiation factors has provided a simple means to assemble active ribosomes on an mRNA that can be used to investigate these downstream stages in the translation process. Here we describe the methods for the assembly of active mammalian ribosomes on the CrPV IGR IRES and for reconstitution and analysis of subsequent steps in the elongation process. The composition of the reconstituted in vitro translation system can be fully controlled, and we therefore suggest that the methods described here could in future be adapted to permit template-dependent synthesis of peptidomimetics by eukaryotic ribosomes, by reassigning individual codons in an mRNA to non-natural amino acids using tRNAs that have been appropriately mischarged either chemically or enzymatically.     

New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation.

We recently demonstrated that the very long 5'-untranslated region (5'-UTR) of the vascular endothelial growth factor (VEGF) mRNA contains two independent internal ribosome entry sites (IRES A and B). In the human sequence, four potential CUG translation initiation codons are located in between these IRES and are in frame with the classical AUG start codon. By in vitro translation and COS-7 cell transfections, we demonstrate that a high mol wt VEGF isoform [called large VEGF (L-VEGF)] is generated by an alternative translation initiation process, which occurs at the first of these CUG codons. Using a bicistronic strategy, we show that the upstream IRES B controls the translation initiation of L-VEGF. This isoform is 206 amino acids longer than the classical AUG-initiated form. With a specific antibody raised against this NH2 extension, we show that the L-VEGF is present in different mouse tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH2-specific fragment and a fragment with an apparent size similar to that of the classical AUG-initiated form. This cleavage requires the integrity of a hydrophobic sequence located in the central part of the L-VEGF molecule. This sequence actually plays the role of signal peptide in the classical AUG-initiated form. The AUG-initiated form and the COOH cleavage product of the L-VEGF are both secreted. In contrast, the large isoform and its NH2 fragment present an intracellular localization. These data unravel a further level of complexity in the regulation of VEGF expression.     

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λ c I and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.