Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Thermodynamically Favorable Interactions between eIF4E Binding Domain of eIF4GI with Structured 5'-Untranslated Regions Drive Cap-Independent Translation of Selected mRNAs.

During cellular stress conditions, particularly those seen in multiple cancers, canonical cap-dependent translation is suppressed and a subset of cellular mRNAs (e.g., those encoding FGF-9, HIF-1α, and p53, among others) is known to translate in a cap-independent manner. Human eIF4GI specifically binds to the highly structured 5'-untranslated regions (5'UTRs) of these mRNAs to promote cap-independent translation. The thermodynamics of these protein-RNA interactions have not been explored, and such information will aid in understanding the basic interactions and in potential design of therapeutic drugs. Using fluorescence quenching-based assays and site-directed mutagenesis, we determined the thermodynamic properties of three eIF4GI constructs binding to the 5'UTRs of FGF-9, HIF-1α, and p53 mRNA. These three constructs were designed to explore the importance of the eIF4E binding domain of eIF4GI, which has been shown to be important in binding and selectivity. eIF4GI557-1599, containing the eIF4E binding domain, had higher binding enthalpy (-21 to -14 kJ mol-1 higher), suggesting increased hydrogen bonding, whereas for eIF4GI682-1599 lacking the eIF4E binding domain, binding was entropically favored (TΔS/ΔG of 46-85%), suggesting hydrophobic forces and/or less specific binding. A third construct where a cluster of positively charged amino acids was changed to neutral amino acids showed intermediate properties. Circular dichroism spectra confirmed the significant role of eIF4E binding domain in stable bond formation between eIF4GI and mRNAs via conformational changes. Together, these data contribute to a better understanding of the molecular forces involved in eIF4GI-mRNA recognition and elucidate properties important for the design of small molecules to mediate these interactions.

5'-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs.

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

Eukaryotic initiation factor (eIF) 3 mediates Barley Yellow Dwarf Viral mRNA 3'-5' UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation.

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA virus that lacks the canonical 5' 7-methylguanosine cap and a 3' poly-A tail. Instead, BYDV utilizes a cruciform cap independent translation element (CITE) in its 3'UTR RNA (BYDV-like CITE or BTE) that binds eukaryotic translation initiation factor (eIF) 4F and recruits 40S ribosomal subunits in the presence of active helicase factors (eIF4A, eIF4B, eIF4F and ATP). A long-range, 5-nucleotide, base-pairing kissing loop interaction between the 3'BTE and a 5'UTR stem-loop is necessary for translation to initiate. The 40S-eIF complex does not bind to the BYDV 5'UTR, suggesting the involvement of additional factors. We identified eIF3 as a component of the 3'BTE recruited complex using affinity-tagged 3'BTE RNA pull-down assays. Fluorescence anisotropy binding and gel shift assays showed that the 3'BTE and 5'UTR RNAs can simultaneously and non-competitively bind eIF3 in the presence of active helicase factors forming a single, macromolecular complex. Further, quantitative studies showed eIF3 increased recruitment of the 40S subunit by more than 25-fold. We propose a new role for eIF3, where eIF3 bridges BYDV's UTRs, stabilizes the long-range 5'-3' interaction, and facilitates recruitment of the 40S-eIF complex to the 5'UTR, leading to translation initiation.

The 3' mRNA I-shaped structure of maize necrotic streak virus binds to eukaryotic translation factors for eIF4F-mediated translation initiation.

Unlike the mRNAs of their eukaryotic hosts, many RNAs of viruses lack a 5' m7GpppN cap and the 3' polyadenosine tail, and yet they are translated efficiently. Plant RNA viruses, in particular, have complex structures within their mRNA UTRs that allow them to bypass some cellular translation control steps. In the 3' UTR of maize necrotic streak virus (MNeSV), an I-shaped RNA structure (ISS) has been shown to bind eukaryotic initiation factor (eIF)4F and to mediate viral translation initiation. A 5'-3' RNA "kissing-loop" interaction is required for optimal translation. However, the details of how the 3' ISS mediates translation initiation are not well understood. Here, we studied the binding of the 3' ISS with eIFs. The eIF4A-eIF4B complex was found to increase binding affinity of eIF4F with the 3' ISS by 4-fold (from KD = 173 ± 34 nm to KD = 48 ± 11 nm). Pre-steady-state analysis indicated that the eIF4A-eIF4B complex increased the RNA association rate and decreased the dissociation rate in an ATP-independent manner. Furthermore, our findings suggest that eIF4F could promote binding of the 3' ISS with the MNeSV 5'UTR, enhancing the long-distance kissing-loop interaction. However, the association of the 5'UTR with the 3' ISS-eIF4F complex did not increase 40S ribosomal subunit binding affinity. These quantitative results suggest a stepwise model in which the first committed step is eIF4F binding to the 3' ISS, followed by an interaction with the 5'UTR and subsequent 40S ribosomal subunit binding.

Eukaryotic translation initiation factor 4G (eIF4G) coordinates interactions with eIF4A, eIF4B, and eIF4E in binding and translation of the barley yellow dwarf virus 3' cap-independent translation element (BTE).

Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation.

Recruitment of the 40S ribosome subunit to the 3'-untranslated region (UTR) of a viral mRNA, via the eIF4 complex, facilitates cap-independent translation.

Barley yellow dwarf virus mRNA, which lacks both cap and poly(A) tail, has a translation element (3'-BTE) in its 3'-UTR essential for efficient translation initiation at the 5'-proximal AUG. This mechanism requires eukaryotic initiation factor 4G (eIF4G), subunit of heterodimer eIF4F (plant eIF4F lacks eIF4A), and 3'-BTE-5'-UTR interaction. Using fluorescence anisotropy, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) analysis, and toeprinting, we found that (i) 40S subunits bind to BTE (Kd = 350 ± 30 nm), (ii) the helicase complex eIF4F-eIF4A-eIF4B-ATP increases 40S subunit binding (Kd = 120 ± 10 nm) to the conserved stem-loop I of the 3'-BTE by exposing more unpaired bases, and (iii) long distance base pairing transfers this complex to the 5'-end of the mRNA, where translation initiates. Although 3'-5' interactions have been recognized as important in mRNA translation, barley yellow dwarf virus employs a novel mechanism utilizing the 3'-UTR as the primary site of ribosome recruitment.

Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.

Eukaryotic initiation factor (eIF) 4F binding to barley yellow dwarf virus (BYDV) 3'-untranslated region correlates with translation efficiency.

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3'-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the "on" rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3' BTE with higher affinity than for either m(7)G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3' BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation.

Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genome-linked protein (VPg).

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.

Modeling the Structure and DAP5 Binding Site of a Cap-Independent Translational Enhancer mRNA.

Cap-independent translation initiation in eukaryotes involves initiation factor (eIF) binding to a transcript's 5' untranslated region (UTR). Internal-ribosome-entry-site (IRES)-like cap-independent translation initiation does not require a free 5' end for eIF binding, as eIFs recruit the ribosome to or near the start codon. For viral mRNA, recruitment usually utilizes RNA structure, such as a pseudoknot. However, for cellular mRNA cap-independent translation, no consensus RNA structures or sequences have yet been identified for eIF binding. Fibroblast-growth factor 9 (FGF-9) is a member of a subset of mRNA that are cap-independently upregulated in breast and colorectal cancer cells using this IRES-like method. Death-associated factor 5 (DAP5), an eIF4GI homolog, binds directly to the FGF-9 5' UTR to initiate translation. However, the DAP5 binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which need a free 5' end to stimulate cap-independent translation. We propose that a particular RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5 binding site. Using SHAPE-seq, we modeled the FGF-9 5' UTR RNA's complex secondary and tertiary structure in vitro. Further, DAP5 footprinting and toeprinting experiments show DAP5's preference for one face of this structure. DAP5 binding appears to stabilize a higher-energy RNA fold that frees the 5' end to solvent and brings the start codon close to the recruited ribosome. Our findings offer a fresh perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Poly(A)-binding protein increases the binding affinity and kinetic rates of interaction of viral protein linked to genome with translation initiation factors eIFiso4F and eIFiso4F·4B complex.

VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem.283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K(d) = 24.3 ± 1.6 nM) as compared to that with eIFiso4F alone (K(d) = 81.3 ± 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F·4B·PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 ± 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 ± 1.5 s(-1)) . The dissociation rate was 3-fold slower (k(-2) = 6.5 ± 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 ± 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 ± 3.0 to 44.0 ± 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.

Iron responsive mRNAs: a family of Fe2+ sensitive riboregulators.

Messenger RNAs (mRNAs) are emerging as prime targets for small-molecule drugs. They afford an opportunity to assert control over an enormous range of biological processes: mRNAs regulate protein synthesis rates, have specific 3-D regulatory structures, and, in nucleated cells, are separated from DNA in space and time. All of the many steps between DNA copying (transcription) and ribosome binding (translation) represent potential control points. Messenger RNAs can fold into complex, 3-D shapes, such as tRNAs and rRNAs, providing an added dimension to the 2-D RNA structure (base pairing) targeted in many mRNA interference approaches. In this Account, we describe the structural and functional properties of the IRE (iron-responsive element) family, one of the few 3-D mRNA regulatory elements with known 3-D structure. This family of related base sequences regulates the mRNAs that encode proteins for iron metabolism. We begin by considering the IRE-RNA structure, which consists of a short (~30-nucleotide) RNA helix. Nature tuned the structure by combining a conserved AGU pseudotriloop, a closing C-G base pair, and a bulge C with various RNA helix base pairs. The result is a set of IRE-mRNAs with individual iron responses. The physiological iron signal is hexahydrated ferrous ion; in vivo iron responses vary over 10-fold depending on the individual IRE-RNA structure. We then discuss the interaction between the IRE-RNA structure and the proteins associated with it. IRE-RNA structures, which are usually noncoding, tightly bind specific proteins called IRPs. These repressor proteins are bound to IRE-RNA through C-bulge and AGU contacts that flip out a loop AG and a bulge C, bending the RNA helix. After binding, the exposed RNA surface then invites further interactions, such as with iron and other proteins. Binding of the IRE-RNA and the IRP also changes the IRP conformation. IRP binding stabilities vary 10-fold within the IRE family, reflecting individual IRE-RNA paired and unpaired bases. This variation contributes to the graded (hierarchical) iron responses in vivo. We also consider the mechanisms of IRE-mRNA control. The binding of Fe(2+) to IRE-RNA facilitates IRP release and the binding of eukaryotic initiation factors (eIFs), which are proteins that assemble mRNA, ribosomes, and tRNA for translation. IRE-RNAs are riboregulators for the inorganic metabolic signal, Fe(2+); they control protein synthesis rates by changing the distribution of the iron metabolic mRNAs between complexes with enhancing eIFs and inhibitory IRPs. The regulation of mRNA in the cytoplasm of eukaryotic cells is a burgeoning frontier in biomedicine. The evolutionarily refined IRE-RNAs, although absent in plants and bacteria, constitute a model system for 3-D mRNAs in all organisms. IRE-mRNAs have yielded "proof of principle" data for small-molecule targeting of mRNA structures, demonstrating tremendous potential for chemical manipulation of mRNA and protein synthesis in living systems.

Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F, eIF4B and PABP.

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A)(20) increases the binding affinity of eIF4F·4B and eIF4F·PABP complexes to IRES RNA ~2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~11-fold with the addition of PABP, eIF4B, and poly(A)(20) together. Whereas, poly(A)(20) alone increases the binding affinity of eIF4F·4B·PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A)(20) was enthalpy-driven and entropy-favorable. Poly(A)(20) decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F·4B·PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F·4B·PABP with IRES-RNA.

Kinetic analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA.

Myc, Mad, and Max proteins belong to the basic helix-loop-helix leucine zipper family of transcription factors. They bind to a specific hexanucleotide element of DNA, the E-box (CACGTG). To be biologically active, Myc and Mad require dimerization with Max. For the route of complex assembly of these dimers, there are two proposed pathways. In the monomer pathway, two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. In the dimer pathway, two monomers form a dimer first prior to association with DNA. The monomer pathway is kinetically favored. In this report, stopped-flow polarization was utilized to determine the rates and temperature dependence of all of the individual steps for both assembly pathways. Myc.Max dimerization had a rate constant approximately 5- and approximately 2-fold higher than those of Max.Max and Mad.Max dimerization, respectively. The protein dimerization rates as well as the dimer-DNA rates were found to be independent of concentration, suggesting conformational changes were rate-limiting. The Arrhenius activation energies for the dimerization of Myc, Mad, and Max with Max were 20.4 +/- 0.8, 29 +/- 0.6, and 40 +/- 0.2 kJ/mol, respectively. Further, rate constants for Max.Max homodimer DNA binding are significantly higher than for Myc.Max and Mad.Max heterodimers binding to DNA. Monomer-DNA binding showed a faster rate than dimer-DNA binding. These studies show the rate-limiting step for the dimer pathway is the formation of protein dimers, and this reaction is slower than formation of protein dimers on the DNA interface, kinetically favoring the monomer pathway.

Kinetic mechanism for the binding of eIF4F and tobacco Etch virus internal ribosome entry site rna: effects of eIF4B and poly(A)-binding protein.

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.

Direct Fe2+ sensing by iron-responsive messenger RNA:repressor complexes weakens binding.

Fe(2+) is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the K(d) value increases 17-fold in 5'-untranslated region IRE-RNA:repressor complexes; Fe(2+), is studied in the absence of O(2). Other metal ions, Mn(2+) and Mg(2+) have similar effects to Fe(2+) but the required Mg(2+) concentration is 100 times greater than for Fe(2+) or Mn(2+). Metal ions also weaken ethidium bromide binding to IRE-RNA with no effect on IRP fluorescence, using Mn(2+) as an O(2)-resistant surrogate for Fe(2+), indicating that metal ions bound IRE-RNA but not IRP: Fe(2+) decreases IRP repressor complex stability of ferritin IRE-RNA 5-10 times compared with 2-5 times for mitochondrial aconitase IRE-RNA, over the same concentration range, suggesting that differences among IRE-RNA structures contribute to the differences in the iron responses observed in vivo. The results show the IRE-RNA:repressor complex literally responds to Fe(2+), selectively for each IRE-mRNA.

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G.

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.

Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F.4B with internal ribosome entry site (IRES) of tobacco etch virus RNA.

In wheat germ, the interaction between poly(A)-binding protein and eukaryotic initiation factor eIF 4G increases the affinity of eIF4E for the cap by 20-40-fold. Recent findings that wheat germ eIF4G is required for interaction with the IRES, pseudoknot 1 (PK1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of PABP on the interaction of eIF4F with PK1. The fluorescence anisotropy data showed addition of PABP to eIF4F increased the binding affinity approximately 2.0-fold for PK1 RNA as compared with eIF4F alone. Addition of both PABP and eIF4B to eIF4F enhance binding affinity to PK1 about 4-fold, showing an additive effect rather than the large increase in affinity shown for cap binding. The van't Hoff analyses showed that PK1 RNA binding to eIF4F, eIF4F.PABP, eIF4F.4B and eIF4F.4B.PABP is enthalpy-driven and entropy-favorable. PABP and eIF4B decreased the entropic contribution 65% for binding of PK1 RNA to eIF4F. The lowering of entropy for the formation of eIF4F.4B.PABP-PK1 complex suggested reduced hydrophobic interactions for complex formation. Overall, these results demonstrate the first direct effect of PABP on the interaction of eIF4F and eIF4F.4B with PK1 RNA.