Stochastic scanning events on the GCN4 mRNA 5' untranslated region generate cell-to-cell heterogeneity in the yeast nutritional stress response.

Gene expression stochasticity is inherent in the functional properties and evolution of biological systems, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. In a distinct form of non-transcriptional noise, we find that interactions of the yeast translation machinery with the GCN4 mRNA 5'UTR, which underpins starvation-induced regulation of this transcriptional activator gene, manifest stochastic variation across cellular populations. We use flow cytometry, fluorescence-activated cell sorting and microfluidics coupled to fluorescence microscopy to characterize the cell-to-cell heterogeneity of GCN4-5'UTR-mediated translation initiation. GCN4-5'UTR-mediated translation is generally not de-repressed under non-starvation conditions; however, a sub-population of cells consistently manifests a stochastically enhanced GCN4 translation (SETGCN4) state that depends on the integrity of the GCN4 uORFs. This sub-population is eliminated upon deletion of the Gcn2 kinase that phosphorylates eIF2α under nutrient-limitation conditions, or upon mutation to Ala of the Gcn2 kinase target site, eIF2α-Ser51. SETGCN4 cells isolated using cell sorting spontaneously regenerate the full bimodal population distribution upon further growth. Analysis of ADE8::ymRuby3/ GCN4::yEGFP cells reveals enhanced Gcn4-activated biosynthetic pathway activity in SETGCN4 cells under non-starvation conditions. Computational modeling interprets our experimental observations in terms of a novel translational noise mechanism underpinned by natural variations in Gcn2 kinase activity.

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

Translation initiation events on structured eukaryotic mRNAs generate gene expression noise.

Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem-loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem-loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem-loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise.

Minimum-noise production of translation factor eIF4G maps to a mechanistically determined optimal rate control window for protein synthesis.

Gene expression noise influences organism evolution and fitness. The mechanisms determining the relationship between stochasticity and the functional role of translation machinery components are critical to viability. eIF4G is an essential translation factor that exerts strong control over protein synthesis. We observe an asymmetric, approximately bell-shaped, relationship between the average intracellular abundance of eIF4G and rates of cell population growth and global mRNA translation, with peak rates occurring at normal physiological abundance. This relationship fits a computational model in which eIF4G is at the core of a multi-component-complex assembly pathway. This model also correctly predicts a plateau-like response of translation to super-physiological increases in abundance of the other cap-complex factors, eIF4E and eIF4A. Engineered changes in eIF4G abundance amplify noise, demonstrating that minimum stochasticity coincides with physiological abundance of this factor. Noise is not increased when eIF4E is overproduced. Plasmid-mediated synthesis of eIF4G imposes increased global gene expression stochasticity and reduced viability because the intrinsic noise for this factor influences total cellular gene noise. The naturally evolved eIF4G gene expression noise minimum maps within the optimal activity zone dictated by eIF4G's mechanistic role. Rate control and noise are therefore interdependent and have co-evolved to share an optimal physiological abundance point.

Found in translation: another RNA helicase function.

In a recent issue of Cell, Pisareva et al. (2008) reveal that DHX29, a previously uncharacterized mammalian DExH-box protein, facilitates translation initiation on mRNAs with structured 5' untranslated regions.

Rate control in yeast protein synthesis at the population and single-cell levels.

Yeast commits approximately 76% of its energy budget to protein synthesis and the efficiency and control of this process are accordingly critical to organism growth and fitness. We now have detailed genetic, biochemical and biophysical knowledge of the components of the eukaryotic translation machinery. However, these kinds of information do not, in themselves, give us a satisfactory picture of how the overall system is controlled. This is where quantitative system analysis can enable a step-change in our understanding of biological resource management and how this relates to cell physiology and evolution. An important aspect of this more system-oriented approach to translational control is the inherent heterogeneity of cell populations that is generated by gene expression noise. In this short review, we address the fact that, although the vast majority of our knowledge of the translation machinery is based on experimental analysis of samples that each contain hundreds of millions of cells, in reality every cell is unique in terms of its composition and control properties. We have entered a new era in which research into the heterogeneity of cell systems promises to provide answers to many (previously unanswerable) questions about cell physiology and evolution.

Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes.

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins.

Local control of a disorder-order transition in 4E-BP1 underpins regulation of translation via eIF4E.

The molecular mechanism underpinning regulation of eukaryotic translation initiation factor eIF4E by 4E-BP1 has remained unclear. We use isothermal calorimetry, circular dichroism, NMR, and computational modeling to analyze how the structure of the eIF4E-binding domain of 4E-BP1 determines its affinity for the dorsal face of eIF4E and thus the ability of this regulator to act as a competitive inhibitor. This work identifies the key role of solvent-facing amino acids in 4E-BP1 that are not directly engaged in interactions with eIF4E. These amino acid residues influence the propensity of the natively unfolded binding motif to fold into a conformation, including a stretch of α-helix, that is required for tight binding to eIF4E. In so doing, they contribute to a free energy landscape for 4E-BP1 folding that is poised so that phosphorylation of S65 at the C-terminal end of the helical region can modulate the propensity of folding, and thus regulate the overall free energy of 4E-BP1 binding to eIF4E, over a physiologically significant range. Thus, phosphorylation acts as an intramolecular structural modulator that biases the free energy landscape for the disorder-order transition of 4E-BP1 by destabilizing the α-helix to favor the unfolded form that cannot bind eIF4E. This type of order-disorder regulatory mechanism is likely to be relevant to other intermolecular regulatory phenomena in the cell.

Reengineering orthogonally selective riboswitches.

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

Translation initiation: variations in the mechanism can be anticipated.

Translation initiation is a critical step in protein synthesis. Previously, two major mechanisms of initiation were considered as essential: prokaryotic, based on SD interaction; and eukaryotic, requiring cap structure and ribosomal scanning. Although discovered decades ago, cap-independent translation has recently been acknowledged as a widely spread mechanism in viruses, which may take place in some cellular mRNA translations. Moreover, it has become evident that translation can be initiated on the leaderless mRNA in all three domains of life. New findings demonstrate that other distinguishable types of initiation exist, including SD-independent in Bacteria and Archaea, and various modifications of 5' end-dependent and internal initiation mechanisms in Eukarya. Since translation initiation has developed through the loss, acquisition, and modification of functional elements, all of which have been elevated by competition with viral translation in a large number of organisms of different complexity, more variation in initiation mechanisms can be anticipated.

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Elucidating mechanistic principles underpinning eukaryotic translation initiation using quantitative fluorescence methods.

Eukaryotic translation initiation is an intricate process involving at least 11 formally classified eIFs (eukaryotic initiation factors), which, together with the ribosome, comprise one of the largest molecular machines in the cell. Studying such huge macromolecular complexes presents many challenges which cannot readily be overcome by traditional molecular and structural methods. Increasingly, novel quantitative techniques are being used to further dissect such complex assembly pathways. One area of methodology involves the labelling of ribosomal subunits and/or eIFs with fluorophores and the use of techniques such as FRET (Förster resonance energy transfer) and FA (fluorescence anisotropy). The applicability of such techniques in such a complex system has been greatly enhanced by recent methodological developments. In the present mini-review, we introduce these quantitative fluorescence methods and discuss the impact they are beginning to have on the field.

Multisite rate control analysis identifies ribosomal scanning as the sole high-capacity/low-flux-control step in mRNA translation.

Control of complex intracellular pathways such as protein synthesis is critical to organism survival, but is poorly understood. Translation of a reading frame in eukaryotic mRNA is preceded by a scanning process in which a subset of translation factors helps guide ribosomes to the start codon. Here, we perform comparative analysis of the control status of this scanning step that sits between recruitment of the small ribosomal subunit to the m7 GpppG-capped 5'end of mRNA and of the control exerted by downstream phases of polypeptide initiation, elongation and termination. We have utilized a detailed predictive model as guidance for designing quantitative experimental interrogation of control in the yeast translation initiation pathway. We have built a synthetic orthogonal copper-responsive regulatory promoter (PCuR3 ) that is used here together with the tet07 regulatory system in a novel dual-site in vivo rate control analysis strategy. Combining this two-site strategy with calibrated mass spectrometry to determine translation factor abundance values, we have tested model-based predictions of rate control properties of the in vivo system. We conclude from the results that the components of the translation machinery that promote scanning collectively function as a low-flux-control system with a capacity to transfer ribosomes into the core process of polypeptide production that exceeds the respective capacities of the steps of polypeptide initiation, elongation and termination. In contrast, the step immediately prior to scanning, that is, ribosome recruitment via the mRNA 5' cap-binding complex, is a high-flux-control step.

The mRNA cap-binding protein eIF4E in post-transcriptional gene expression.

Eukaryotic initiation factor 4E (eIF4E) has central roles in the control of several aspects of post-transcriptional gene expression and thereby affects developmental processes. It is also implicated in human diseases. This review explores the relationship between structural, biochemical and biophysical aspects of eIF4E and its function in vivo, including both long-established roles in translation and newly emerging ones in nuclear export and mRNA decay pathways.

Distributed control for recruitment, scanning and subunit joining steps of translation initiation.

Protein synthesis utilizes a large proportion of the available free energy in the eukaryotic cell and must be precisely controlled, yet up to now there has been no systematic rate control analysis of the in vivo process. We now present a novel study of rate control by eukaryotic translation initiation factors (eIFs) using yeast strains in which chromosomal eIF genes have been placed under the control of the tetO7 promoter system. The results reveal that, contrary to previously published reports, control of the initiation pathway is distributed over all of the eIFs, whereby rate control (the magnitude of their respective component control coefficients) follows the order: eIF4G > eIF1A > eIF4E > eIF5B. The apparent rate control effects of eIFs observed in standard cell-free extract experiments, on the other hand, do not accurately reflect the steady state in vivo data. Overall, this work establishes the first quantitative control framework for the study of in vivo eukaryotic translation.

Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways.

Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

Biophysical studies of the translation initiation pathway with immobilized mRNA analogs.

A growing number of biophysical techniques use immobilized reactants for the quantitative study of macromolecular reactions. Examples of such approaches include surface plasmon resonance, atomic force microscopy, total reflection fluorescence microscopy, and others. Some of these methods have already been adapted for work with immobilized RNAs, thus making them available for the study of many reactions relevant to translation. Published examples include the study of kinetic parameters of protein/RNA interactions and the effect of helicases on RNA secondary structure. The common denominator of all of these techniques is the necessity to immobilize RNA molecules in a functional state on solid supports. In this chapter, we describe a number of approaches by which such immobilization can be achieved, followed by two specific examples for applications that use immobilized RNAs.

Dcs2, a novel stress-induced modulator of m7GpppX pyrophosphatase activity that locates to P bodies.

The eukaryotic "scavenger" type decapping enzyme, an m(7)GpppX pyrophosphatase, is active in cellular mRNA metabolism and thereby influences posttranscriptional gene expression. The yeast version of this enzyme, Dcs1, catalyses cleavage of 5'end m(7)G-oligoribonucleotide fragments generated by 3'-->5' exonucleolytic decay, and cleavage of m(7)GDP generated by Dcp1/Dcp2-mediated decapping in the 5'-->3' decay pathway. We show that Dcs1 is active as a homodimer with low KM values for cleavage of m(7)GpppG (0.14 microM) and m(7)GDP (0.26 microM). Previous work showed that the paralogous DCS2 gene is transcriptionally induced via the amp-PKA pathway as yeast enters diauxie. The resulting Dcs2 protein forms a heterodimer together with Dcs1, both modulating Dcs1 substrate specificity and suppressing its k(cat). Since Dcs2 is recruited into cytoplasmic P bodies, its inhibitory function may be focused in these centres of mRNA storage/turnover. Dcs2 is therefore a novel type of stress-induced regulatory protein that modulates m(7)GpppX pyrophosphatase activity. Moreover, inhibition of Dcs1 activity by Dcs2, like depletion of Dcs1, reduces chronological life span, possibly by modulating m(7)G misincorporation into nucleic acids. This could potentially link control of mRNA metabolism with senescence.

Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation.

The small (40 S) subunit of the eukaryotic ribosome may have to scan more than 2000 nucleotides (>600 nm) from its 5'cap recruiting point on an mRNA molecule before initiating on a translation start codon. As with many other processes in living cells, including transcription, editing, mRNA splicing, pre-rRNA processing, RNA transport and RNA decay, scanning is facilitated by helicase activity. However, precise quantitative data on the molecular mechanism of scanning, including the roles of helicases, are lacking. Here, we describe a novel atomic force microscopy (AFM)-based procedure to examine the roles of two yeast helicases, eIF4A and Ded1, previously implicated in translation initiation by genetic and biochemical studies. Our results show that eIF4A, especially in the presence of its "cofactor" eIF4B, promotes ATP-dependent unwinding of localised secondary structure in long RNA molecules under tensional loading, albeit only at high protein:RNA ratios. Thus eIF4A can act to separate only a limited number of base-pairs, possibly via a steric unwinding mechanism. In contrast, Ded1 is more effective in reducing (by up to 50 pN at an AFM loading rate of 14 nNs(-1)) the force necessary to disrupt an RNA stem-loop, and thus shows significant kinetic competence to facilitate fast unwinding. These single molecule experiments indicate that Ded1 is likely to act as the more potent unwinding factor on natural mRNA substrates.

Folding transitions during assembly of the eukaryotic mRNA cap-binding complex.

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.