eIF2ß zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2ß subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2ß-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2ßS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2ßS264Y mutation has eIF2ß-γ interaction defect. Consistently, the eIF2ßT238A intragenic suppressor mutation restored the eIF2ß-γ and Met-tRNAiMet binding. The eIF2ß-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2ßT238A mutation, suggesting the eIF2ß-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2ßF217A/Q221A double mutation in the HTH domain. The eIF2ßT238A suppressor mutation could not rescue the eIF2ß binding defect of the eIF2γV281K mutation; however, combining the eIF2ßS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2ß with the eIF2γ subunit via its α1-helix, the eIF2ß-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2ß-γ interacts via two distinct binding sites.

Covalent Organic Frameworks as Nano-Reservoir for Room Temperature RNA Storage.

The emerging role of Ribonucleic acids (RNAs) as therapeutics is alluring. However, RNAs are extremely labile under ambient conditions and typically need to be stored in cryogenic conditions (-20 °C to -80 °C). Hence, storage, stabilization, and transportation of RNA under ambient conditions have been an arduous task and remain an unsolved problem. In this work, a guanidinium-based ionic covalent organic framework (COF), TTGCl with nanotubular morphology, was synthesized and used as nano-reservoirs for room-temperature storage of RNA. To understand the role of the nanotubular morphology and chemical nature of TTGCl in stabilizing the RNA structure and for comparison purposes, a neutral COF, TMT-TT, is synthesized and studied. Further, density functional theory (DFT) studies confirmed non-covalent interaction between the COFs and the RNA nucleobases, facilitating reversible storage of RNA. RNA loaded in COFs was found to be resistant to enzymatic degradation when treated with RNase. Gel electrophoresis and sequencing confirmed the structural integrity of the recovered RNAs and their further processibility.

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage.

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation-reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection.

The 40S ribosome plays a critical role in start codon selection. To gain insights into the role of its 18S rRNA in start codon selection, a suppressor screen was performed that suppressed the preferential UUG start codon recognition (Suppressor of initiation codon: Sui- phenotype) associated with the eIF5G31R mutant. The C1209U mutation in helix h32 of 18S rRNA was found to suppress the Sui- and Gcn- (failure to derepress GCN4 expression) phenotype of the eIF5G31R mutant. The C1209U mutation suppressed Sui- and Gcd- (constitutive derepression of GCN4 expression) phenotype of eIF2ßS264Y , eIF1K60E , and eIF1A-ΔC mutation. We propose that the C1209U mutation in 40S ribosomal may perturb the premature head rotation in 'Closed/PIN ' state and enhance the stringency of translation start site selection.

Fidelity of HIS4 start codon selection influences 3-amino-1,2,4-triazole sensitivity in GTPase activating protein function defective eIF5.

The eIF5 protein plays an important role in the fidelity of AUG start codon selection. However, the hyper GTPase eIF5G31R mutation in yeast causes preferential utilization of UUG as initiation codon and is termed as suppressor of initiation codon (Sui-) phenotype. The eIF5G31R mutant recognizes upUUG initiation codon from the 5' regulatory leader region of GCN4 transcript and dominantly represses GCN4 expression thereby conferring sensitivity to 3-amino-1,2,4-triazole (3AT)-induced starvation. The 3AT sensitivity was rescued by supplementing HIS4UUG allele. The eIF5G31R mutant has a better efficiency of UUG codon recognition from the HIS4UUG allele under starvation conditions. Moreover, the expression of HIS4UUG allele was significantly lower than the critical level causing additional derepression of GCN4 expression in eIF5G31R mutant to rescue its 3AT sensitivity. The overexpression of eIF1 improved expression of HIS4AUG allele and GCN4 transcript causing 3AT resistance, whereas overexpression of eIF1 resulted in diminished UUG codon recognition of HIS4UUG allele causing 3AT sensitivity, despite having higher GCN4 expression. This paper reports the critical role of HIS4 expression necessary in response to 3AT-induced starvation in the eIF5G31R mutant which is ostensibly not a direct target of 3AT inhibition.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui-) phenotype due to its hyper GTPase activity. The eIF5G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn- phenotype) in the eIF5G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF.

Translation initiation factor 2gamma mutant alters start codon selection independent of Met-tRNA binding.

Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNA(i)(Met) and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNA(i)(Met) to the 40S ribosomal subunit, and previous studies identified Sui(-) mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNA(i)(Met) binding. Consistently, an eIF2gamma-N135D GTP-binding domain mutation impairs Met-tRNA(i)(Met) binding and causes a Sui(-) phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNA(i)(Met) binding affinity and cell growth; however, only A208V suppresses the Sui(-) phenotype associated with the eIF2gamma-N135D mutation. An eIF2gamma-A219T mutation impairs Met-tRNA(i)(Met) binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui(-) phenotype associated with the eIF2gamma-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui(-) phenotype of the eIF2gamma mutants. We propose that structural alterations in eIF2gamma subtly alter the conformation of Met-tRNA(i)(Met) on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA(i)(Met) binding affinity.

Secretory and GM1 receptor binding role of N-terminal region of LTB in Vibrio cholerae.

Heat labile enterotoxin from enterotoxigenic Escherichia coli is similar to cholera toxin (CT) and is a leading cause of diarrhea in developing countries. It consists of an enzymatically active A subunit (LTA) and a carrier pentameric B subunit (LTB). In the current study, we evaluated the importance of the N-terminal region of LTB by mutation analysis. Deletion of the glutamine (DeltaQ3) residue and a substitution mutation E7G in the alpha1 helix region led to defects in LTB protein secretion. Deletion of the proline residue (DeltaP2) caused a decrease in alpha helicity. The DeltaP2 mutant affected GM(1) ganglioside receptor binding activity without affecting LTB pentamer formation. Upon refolding/reassembly, the DeltaP2 mutant showed defective biological activity. The single substitution mutation (E7D) strengthened the helix, imparting structural stability and thereby improved the GM(1) ganglioside receptor binding activity. Our results demonstrate the important role of N-terminal alpha1 helix in maintaining the structural stability and the integrity of GM(1) ganglioside receptor binding activity.

Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.

Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the alpha1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the alpha1 helix also resulted in loss of expression. However, a single amino acid deletion outside the alpha1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcription-translation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.

Direct binding of translation initiation factor eIF2gamma-G domain to its GTPase-activating and GDP-GTP exchange factors eIF5 and eIF2B epsilon.

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.