Translation Elongation

During translation elongation, the peptidyltransferase reaction (the reaction by which amino acid residues are attached to each other to form proteins) is catalyzed by the rRNA itself.

From: Cell Biology (Third Edition) , 2017

Elongation Factors: Translation☆

D. Hughes , in Reference Module in Life Sciences, 2017

Abstract

Translation elongation factors perform critical functions in protein synthesis in all domains of life, including the delivery of aminoacyl-tRNAs into the ribosome, and the translocation of peptidyl-tRNA from the ribosomal A-site to the ribosomal P-site. Elongation factor Tu (EF-Tu, EF1-alpha) is a GTP-binding protein that is responsible for carrying each aminoacyl-tRNA to the ribosome, a process that involves hydrolysis of GTP and dissociation of the complex. EF-Ts is responsible for exchanging GDP for GTP on EF-Tu, ensuring that EF-Tu is reactivated for aminoacyl-tRNA binding after interacting with the ribosome. A third factor, EF-G (EF2 in eukaryotes), is responsible for catalyzing the translocation of peptidyl-tRNA from the A-site to the P-site. The elongation factors EF-Tu and EF-G are targets for several natural antibiotics, including fusidic acid (targets EF-G) which is used clinically to treat infections by Staphylococcus aureus.

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Improved analyses of regulatory genome, transcriptome and gene function, mutation penetrance, and clinical applications

Moyra Smith , in Progress in Genomic Medicine, 2022

7.15 Translation of mRNA to proteins and associated defects leading to disease

Steps in translation of mRNA to proteins were reviewed by Gabut et al. (2020). Early steps include passage and coupling of the 5′ mRNA regions to the 40s ribosome through action of the eif4 complex that merges with a bound preinitiation complex (PIC). The PIC includes GTP (Guanosine triphosphate), EIF3, EIF5, EIF1 subunits, and EIF1A that together with methionyl initiator TRNA bind to the 40s ribosome unit. The PIC complex was noted to scan the mRNA to identify the AUG initiation codon. Subsequent release of GTP (guanosine triphosphate) and bound initiation factors allow for recruitment and binding of EIF3B and the 80s ribosome unit. The aminoacyl TRNA binding site is exposed, and translation can begin.

7.15.1 Aminoacyl tRNA synthases

Translation elongation requires specific aminoacyl TRNAs being escorted to the ribosome by GTP-coupled elongation factor. Elongation requires movement along the ribosome-coupled mRNA, three nucleotides at a time to add aminoacids that have been bound to TRNAs. A specific aminoacyl tRNA synthetase must select the correct amino acid, and it must select the correct tRNA from the TRNA pool. Fuchs et al. (2019) reviewed aminoacyl tRNA synthetase defects noting that they were particularly encountered in children with psychomotor retardation and seizures.

7.15.2 Noncanonical functions of aminoacyl tRNA synthetases

Musier-Forsyth (2019) reported that the canonical function of aminoacyl tRNA synthetases is well-known and is attachment of cognate tRNAs to specific aminoacids. Additional noncanonical functions have been discovered. Those include regulation or splicing, stimulation of MTOR activity, and DNA repair.

Rubio Gomez and Ibba (2020) reviewed aminoacyl tRNA synthetases and noted that they are not only essential for accurate translation of the genetic code, but they had roles in other processes.

In their primary function, aminoacyl tRNA synthetases were noted to catalyze a two-step reaction. This involved esterification of aminoacid and hydrolysis of ATP to generate aminoacyl tRNA.

Aminoacyl tRNA synthetase defects have been particularly associated with nervous system disorders.

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Translation Elongation in Eukaryotes

William C. Merrick , Anton A. Komar , in Encyclopedia of Biological Chemistry, 2004

The Other Elongation Factor, eEF3

A translation elongation factor unique to yeast and fungi is eEF3. This protein, which contains two-nucleotide-binding sites, appears to be required for the nucleotide-dependent release of the nonacylated tRNA from the ribosomal E site. As this protein is an essential gene product in yeast, it is surprising that an equivalent activity has not been identified in other eukaryotes. However, it has been noted in vitro that only elongation reactions using yeast ribosomes demonstrate the eEF3 requirement, and thus this requirement for eEF3 would appear to reflect unusual properties of the yeast ribosome compared to other eukaryotic ribosomes.

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Non-Conventional Yeast Species for Recombinant Protein and Metabolite Production

Hoang D. Do , ... Chrispian W. Theron , in Reference Module in Life Sciences, 2019

3.2.3 The expression cassette: Emphasis on promoters

3.2.3.1 Constitutive promoters

The promoter of the translation elongation factor-1α ( TEF1) gene (Müller et al., 1998), is the most widely used constitutive promoter in Y. lipolytica (Madzak and Beckerich, 2013). It is regarded as the strongest identified natural promoter for this yeast and hence serves a reference promoter for comparison of strength to other promoters. Another constitutive promoter described for this yeast is the promoter for ribosomal protein S7 (RPS7), which provides more moderate expression levels (Müller et al., 1998).

3.2.3.2 Inducible promoters

The inducible promoters described so far for Y. lipolytica are generally related to the metabolism of carbon sources. Exceptions include the promoter from the XPR2 gene, encoding an alkaline extracellular protease, which is of historical importance since it was the first to be characterized (Ogrydziak and Scharf, 1982) and subsequently used for protein production (Madzak and Beckerich, 2013). However, the demanding conditions to achieve complete induction (high peptide concentrations and a pH above 6), are often unrealistic at an industrial scale, which stimulated the search for other inducible promoters. The only other promoter that has been described as inducible by a compound that is not specifically carbon source is the promoter of the gene encoding β-Isopropylmalate dehydrogenase (LEU2) involved in the biosynthesis of leucine, which is consistently inducible by leucine precursor (Gaillardin and Ribet, 1987).

Due to the lipophilic nature of Y. lipolytica, it makes sense that the pathways of fatty acid metabolism are a major source of inducible promoters. The promoters from the isocitrate lyase (ICL1), acyl-CoA oxidase 2 (POX2) and 3-oxo-acyl-CoA thiolase (POT1) genes are all inducible by hydrophobic substrates (HS) like alkanes, fatty acids and derivates of these compounds, and these were directly compared in a study by Juretzek et al. (2000). pICL1 is additionally also inducible by the water-soluble compounds acetate and ethanol. Another HS-inducible promoter is that of lipase 2 (LIP2) encoding an extracellular lipase (Fickers et al., 2003b), which was found to exhibit less leaky expression with glucose as sole carbon source than pPOX2, allowing better separation of growth and induction phases when required (Sassi et al., 2016). A drawback to using these types of substrates however, is the immiscibility of these hydrophobic substrates in aqueous-based medium, particularly in large-scale bioreactors. Addition of a co-substrate such as glucose to reduce hydrophobic substrates has been investigated (Sassi et al., 2016).

Promoters inducible by water-miscible substances could therefore be more convenient. The promoter of glycerol-3-phosphate dehydrogenase (G3P) is inducible by glycerol (Juretzek et al., 2000). More recently, a series of promoters that are highly inducible by glucose were characterized, namely the promoters of the fructose-bisphosphate aldolase (FBA1), phosphoglycerate mutase (GPM1), and glyceraldehyde‐3‐phosphate dehydrogenase (TDH1) genes (Hong et al., 2012). Similarly, the promoter of the gene encoding an erythrulose dehydrogenase (EYK1) that involved in erythritol metabolism, has been found to be strongly inducible by erythritol and erythrulose (Trassaert et al., 2017).

Intriguingly, for both inducible (pFBA, Hong et al., 2012) and constitutive (pTEF1, Tai and Stephanopoulos, 2013) promoters, there is evidence that maintaining the presence of an intron of the native gene after the promoter sequence can significantly increase the promoter strength. In the case of pICL, the inclusion of the first intron of the ICL gene upstream of genes of interest allowed expression (Nthangeni et al., 2004), while lower expression levels were observed without it (Theron, unpublished results).

Alongside the study of native promoters, a range of synthetic promoters with shorter size and increased and/or tuneable strength have been developed. Their architecture relies on two components: an enhancer element and a core promoter element (Blazeck et al., 2011). The functional dissection of pXPR2 unveiled an upstream activating sequence (UAS1 XPR2 ) poorly affected by cultivation conditions. One to four copies of UAS1 XPR2 (regarded as the enhancer element) were associated with a minimal LEU2 promoter (used as the core element) to create the first series of synthetic promoters available for Y. lipolytica (Madzak et al., 2000). Among them, hp4d (comprising 4 copies of UAS1 XPR2 ) has been widely used for heterologous protein production (Madzak and Beckerich, 2013), though it was demonstrated to be growth-phase dependant rather than strictly constitutive. Later, up to 32 copies of UAS1B XPR2 , a sequence-optimised derivative of UAS1 XPR2 , were used, with promoter strength correlated with the number of enhancer elements (Blazeck et al., 2011). This synthetic promoter design was then extended to other enhancer and core elements of pTEF, pPOX2, pLEU2 (Blazeck et al., 2013, 2011; Shabbir Hussain et al., 2016). Recently, the first series of synthetic promoters inducible by erythritol based on combinations of UAS of pEYK1, with or without associated copies of UAS1B XPR2 , have demonstrated a strength up to 45-fold superior to pTEF (Trassaert et al., 2017). Further engineering of pEYK1 and pEYD1 led to a variety of variable strength promoters (Park et al., 2019). It should however be noted that promoter strength does not always correlate to better productivity, although it is generally the case (Dulermo et al., 2017).

As mentioned earlier, the choice of a terminator is also important (Curran et al., 2015). Commonly used terminators for Y. lipolytica are the XPR2t and minimal XPR2t, LIP2t and PHO5t (Pignède et al., 2000a). Synthetic terminators, which are shorter and present similar or better performances to their native counterparts, have also been used in this yeast (Curran et al., 2015).

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Ribosomal profiling—Diversity and applications

Sunita Giri , Vijay Kumar , in Emerging Concepts in Ribosome Structure, Biogenesis, and Function, 2021

Ribosome profiling of elongating ribosomes

Growing evidences suggest that the translation elongation rate plays a major role in the cotranslational folding of nascent peptide chains and thus fine-tuning of gene expression. During the elongation phase, ribosomes undergo substantial conformational changes to accommodate incoming aminoacyl-tRNAs and slide along the mRNA template ( Wu et al., 2019). As a reason, mRNAs with slow-moving ribosomes tend to yield better folded and more stable proteins. Moreover, the ribosome elongation rate can also influence the mRNA half-life, thereby regulating gene expression. Ribosome profiling has helped in throwing new insights into the elongation process of translation.

Analysis of elongation: Once the translation has been initiated, the ribosome starts synthesizing protein through the elongation process. Thus ribosome profiling of elongating ribosomes is important for studying differential gene expression, measuring global and local translation elongation rates, and identifying novel genes and gene products. In these experiments, cells are pulse-treated with translation elongation inhibitors to immobilize and faithfully capture elongating ribosomes in their in vivo translational positions prior to cell lysis. Cycloheximide has been widely used in the elongating ribosome profiling studies. However, simple liquid nitrogen freezing as well as other antibiotics such as "emetine" in eukaryotes and "chloramphenicol" in bacteria have also been used. The elongation rate for mammalian cells using ribosome profiling has been estimated at five amino acids per second (Wu et al., 2016).

Analysis of differential gene expression: Knowing changes in gene expression in a cell in response to changing conditions is essential for understanding the genetic determinants of phenotypical behavior. Though microarray and RNA-seq techniques have been extensively used for measuring differential gene expression, the correlation between mRNA and protein levels has been insufficient and inaccurate for predicting protein expression in the cell. This could happen when a ribosome is stalled on an mRNA or translation is limited to upstream open reading frames (uORFs) hindering translation of the main protein product ORF. Further, two transcripts expressed at the same level but of different length would produce a different number of short reads aligning to them, as the number of reads is proportional to the length of the transcript. Since ribosomes broadly translate mRNAs at a similar elongation rate, conversion of the absolute number of footprints into ribosome density is used for estimating translation rates. Besides, the triplet periodicity property of ribosome profiling and the generation of subcodon profiles are also used (Michel and Baranov, 2013). Thus ribosome profiling studies combined with the application of some computational tools on the Ribo-seq and RNA-seq data have been successfully used for getting better insights into the differential translation of transcripts.

Role of tRNA landscapes—The tRNA landscape of cells (including the availability of aminoacyl-tRNAs and specific modifications on tRNAs) is considered to be a major determinant of variation in elongation rate. Not surprisingly, an altered tRNA landscape is frequently observed in malignant cells. In fact, the analysis of the gene-specific elongation velocity index in different cell types has shown that the elongation rates are closely linked to their respective tRNA composition (Lian et al., 2016). Consequently, mRNAs with slow elongation indices generally produce correctly folded and more stable proteins. Thus a reduction in elongation velocity may be necessary for the maintenance of cancer cells. Besides an upregulated tRNAs seen in highly metastatic breast cancer cells could promote the stability and translation of transcripts that are enriched for the corresponding codons (Goodarzi et al., 2016). It means that there could be reshaping of protein expression through modulation of ribosome occupancy and transcript stability. Further, in renal cell carcinoma, there is high ribosome occupancy on proline codons due perhaps to the limiting availability of aminoacylated tRNA for proline (Goodarzi et al., 2016).

Thus ribosome profiling not only can measure tRNA levels, low availability of amino acids in cells, and compensatory mechanisms in tumors, but can also reveal the impact of ribosome elongation on the fate of transcripts and protein expression (Goodarzi et al., 2016; Ingolia et al., 2011; Lian et al., 2016; Loayza-Puch et al., 2016; Rooijers et al., 2013; Wu et al., 2016).

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Photosynthesis | Photosystem II: Assembly and Turnover of the Reaction Center D1 Protein in Plant Chloroplasts☆

Sanna Rantala , ... Eva-Mari Aro , in Encyclopedia of Biological Chemistry (Third Edition), 2021

Regulation of the PSII Assembly

Light is required for efficient translation elongation and accumulation of the D1 protein as well as for synthesis of the CP43 and CP47 core proteins ( Eichacker et al., 1990). In addition to light, regulation of synthesis and assembly of the PSII complex involves the availability of a variety of factors, including the ligation of pigments and cofactors (chlorophyll a, pheophytin, β-carotene, Fe, Mn, and plastoquinone) and the availability of the assembly partners (Klein et al., 1988; Herrin et al., 1992).

The chloroplast-encoded core proteins D1, D2, CP43, and CP47 are cotranslationally inserted into the thylakoid membrane (van Wijk et al., 1995; Zhang et al., 1999). Pigments are thought to bind to these proteins during their cotranslational membrane insertion and concomitant association with assembly partner(s) (Kim et al., 1991). Light is needed for chlorophyll biosynthesis at the level of conversion of protochlorophyllide to chlorophyll a (Apel et al., 1980), but we lack the information on details of pigment binding, even though the importance of this event as a regulative step in the assembly of PSII has been known for a long time. It is clear, however, that the chlorophyll-binding proteins of PSII are stabilized by ligation of pigments (Mullet et al., 1990; Eichacker et al., 1996). Most chlorophyll-binding proteins do not accumulate at all in a nonpigmented form (Paulsen, 2001).

Successful synthesis of the PSII core proteins requires the availability of assembly partners. Synthesis of the D2 protein is dependent on the presence of cyt b559 (Müller and Eichacker, 1999). Furthermore, the cyt b559/D2 subcomplex is indispensable for the synthesis of D1, and the cyt b559/D2/D1 subcomplex, in turn, functions as an assembly partner for CP47 (Rokka et al., 2005). From the PSII core proteins, CP43 seems to be synthesized quite independently.

Moreover, dozens of nuclear-encoded auxiliary protein are required for correct and efficient assembly of PSII (Nickelsen and Rengstl, 2013; Pagliano et al., 2013; Järvi et al., 2015). During the D1 protein translation and insertion into the membrane, the stromal proteins LPA1 (Wei et al., 2010) and HCF136 (Plücken et al., 2002) as well as the lumenal immunophilin CYP38 (Sirpiö et al., 2008) guide the proper folding of the D1 protein. The thylakoid lumen proteins are of utmost importance during the assembly of the PSII core complex and OEC proteins. These proteins include several lumenal peptidyl-prolyl isomerases, OEC protein isoforms and Psb27 (Roose and Pakrasi, 2008). Integration of the LHCII proteins to the membrane is dependent on the presence of the thylakoid membrane protein ALB3 (Schneider et al., 2014).

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Photosystem II: Assembly and Turnover of the D1 Protein

Eira Kanervo , Eva-Mari Aro , in Encyclopedia of Biological Chemistry, 2004

Regulation of the PSII Assembly

Light is required for an efficient translation elongation and accumulation of the D1 protein as well as for synthesis of the CP43 and CP47 core proteins. In addition to light, regulation of synthesis and assembly of the PSII complex involves the availability of a variety of factors, including the ligation of pigments and cofactors (chlorophyll a, pheophytin, β-carotene, Fe, Mn, plastoquinone) and the availability of the assembly partners.

The chloroplast-encoded core proteins D1, D2, CP43, and CP47 are cotranslationally inserted into the thylakoid membrane. Pigments are thought to bind to these proteins during their cotranslational membrane translocation and during the concomitant association of the protein with its assembly partner(s). Light is needed for the chlorophyll biosynthesis at the level of conversion of protochlorophyllide to chlorophyll a. Currently, we lack information on details of the pigment binding, even though the importance of this event as a regulative step in the PSII assembly has been known for a long time. It is clear, however, that the chlorophyll-binding proteins of PSII are stabilized by ligation of pigments. Most chlorophyll-binding proteins do not accumulate at all in a nonpigmented form.

Successful synthesis of the PSII core proteins requires the availability of assembly partners. Synthesis of the D2 protein is dependent on the presence of cyt b559. Furthermore, cyt b559/D2 subcomplex is indispensable for the synthesis of D1, and the cyt b559/D2/D1 subcomplex, in turn, functions as an assembly partner for CP47. From the PSII core proteins, CP43 seems to be synthesized quite independently.

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Radical SAM Enzymes

Min Dong , ... Hening Lin , in Methods in Enzymology, 2018

Abstract

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a C single bondC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe–4S] clusters to generate a 5′-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different Csingle bondS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.

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Elongation Factors

D. Hughes , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Other Factors in Bacteria

Other protein factors are also involved in translation elongation. Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. The gene encoding this protein, efp, has been found throughout the bacteria. The homologous protein in eukaryotes is the initiation factor, eIF5A. EF-P binds to a site on the ribosome located between the binding site for the peptidyl tRNA (P-site) and the exiting tRNA (E-site). The essential role of EF-P in the cell may be to correctly position the initiator aa-tRNA in the ribosomal P-site for the first step of peptide bond formation.

An EF-Tu-like elongation factor (SelB) is required to bring selenocysteinyl-tRNA to the ribosome in response to UGA codons in an appropriate context. The interaction of this specialized ternary complex with the ribosome is via a specific interaction with a structure in mRNA containing the UGA codon. Selenocysteine is an essential amino acid but is required in only a few proteins. SelB-like proteins are also found in the archaea and eukaryotes.

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Translational Control and Fidelity

P.J. Farabaugh , in Encyclopedia of Microbiology (Third Edition), 2009

Translation factor mutations

The final class of suppressors affect the translation elongation factors EF-1A and eEF-1A, and the peptide release factors. Mutant forms of bacterial EF-1A and yeast ( S. cerevisiae) eEF-1A have the same effect of suppressing nonsense or frameshift mutations, or both. The mutations are not clustered in the molecule, and in fact affect either of its two functionally distinct regions, the GTP-binding and the tRNA-binding domains. The exact mechanism of suppression is unclear. Those affecting the GTP-binding domain may affect the kinetics of GTP hydrolysis, which under the kinetic proofreading model would be expected to perturb discrimination between correct and incorrect tRNAs. Those in the tRNA-binding domain may change the interaction between the EF-1A ternary complex and the ribosome, perhaps stabilizing the interaction of noncognate tRNAs, which again under that model would tend to reduce discrimination.

Availability of peptide release factors influence the frequency of nonsense codon readthrough both in vivo and in vitro. Reduction in the effective concentration of release factors tends to allow readthrough, to varying degrees, of all three types of nonsense codons. The amino acid inserted tends to be encoded by a codon that is near in sequence to the nonsense codon. This implies that when the rate of recognition of a nonsense codon is limiting, the ribosome will accept noncognate tRNAs. This is consistent with the idea that ribosomal accuracy is kinetically controlled. In yeast, an epigenetic state can arise in which peptide release factor is limiting. The state is brought on by aggregation of the factor and results in increased nonsense codon readthrough. This situation resembles a prion infection since the condition is heritable, though not genetically encoded, and is an active area of investigation given its clinical significance as a model system.

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