Human eIF5 and eIF1A Compete for Binding to eIF5B.

Eukaryotic translation initiation is a multistep process requiring a number of eukaryotic translation initiation factors (eIFs). Two GTPases play key roles in the process. eIF2 brings the initiator Met-tRNAi to the preinitiation complex (PIC). Upon start codon selection and GTP hydrolysis promoted by the GTPase-activating protein (GAP) eIF5, eIF2-GDP is displaced from Met-tRNAi by eIF5B-GTP and is released in complex with eIF5. eIF5B promotes ribosomal subunit joining, with the help of eIF1A. Upon subunit joining, eIF5B hydrolyzes GTP and is released together with eIF1A. We found that human eIF5 interacts with eIF5B and may help recruit eIF5B to the PIC. An eIF5B-binding motif was identified at the C-terminus of eIF5, similar to that found in eIF1A. Indeed, eIF5 competes with eIF1A for binding and has an ∼100-fold higher affinity for eIF5B. Because eIF5 is the GAP of eIF2, the newly discovered interaction offers a possible mechanism for coordination between the two steps in translation initiation controlled by GTPases: start codon selection and ribosomal subunit joining. Our results indicate that in humans, eIF5B displacing eIF2 from Met-tRNAi upon subunit joining may be coupled to eIF1A displacing eIF5 from eIF5B, allowing the eIF5:eIF2-GDP complex to leave the ribosome.

eIF1A/eIF5B interaction network and its functions in translation initiation complex assembly and remodeling.

Eukaryotic translation initiation is a highly regulated process involving multiple steps, from 43S pre-initiation complex (PIC) assembly, to ribosomal subunit joining. Subunit joining is controlled by the G-protein eukaryotic translation initiation factor 5B (eIF5B). Another protein, eIF1A, is involved in virtually all steps, including subunit joining. The intrinsically disordered eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4). The ribosomal complex undergoes conformational rearrangements at every step of translation initiation; however, the underlying molecular mechanisms are poorly understood. Here we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interaction in eIF1A between the folded domain and eIF1A-CTT; and (iii) an intramolecular interaction between eIF5B-D3 and -D4. The intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the ribosome at different stages of translation initiation. Therefore, our results indicate that the interactions between eIF1A and eIF5B are being continuously rearranged during translation initiation. We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the translation initiation complexes, and the roles in the process played by intrinsically disordered protein segments.

Blood reference materials from macaques infected with variant Creutzfeldt-Jakob disease agent.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. STUDY DESIGN AND METHODS: To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrP(TSE) with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. RESULTS: We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrP(TSE) was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. CONCLUSION: The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.

Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors.

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA , tRNA and tRNA . The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of which can form a noncanonical base pair with geometry similar to that of the canonical U•A or C•G Watson-Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson-Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson-Crick pairing.

Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP(TSE)) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP(TSE) in tissues and blood. Macaque vCJD PrP(TSE) did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP(TSE). The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP(TSE). Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP(TSE) was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP(TSE) demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP(TSE) was more permissive than human PrP(TSE) in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP(TSE) from brains of humans and macaques with vCJD. PrP(TSE) signals were reproducibly detected by Western blot in dilutions through 10⁻¹² of vCJD-infected 10% brain homogenates. This is the first report showing PrP(TSE) from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP(TSE) in vCJD-infected human and macaque blood.

Altered dynamics of DNA bases adjacent to a mismatch: a cue for mismatch recognition by MutS.

The structural deviations as well as the alteration in the dynamics of DNA at mismatch sites are considered to have a crucial role in mismatch recognition followed by its repair utilizing mismatch repair family proteins. To compare the dynamics at a mismatch and a non-mismatch site, we incorporated 2-aminopurine, a fluorescent analogue of adenine next to a G.T mismatch, a C.C mismatch, or an unpaired T, and at several other non-mismatch positions. Rotational diffusion of 2-aminopurine at these locations, monitored by time-resolved fluorescence anisotropy, showed distinct differences in the dynamics. This alteration in the motional dynamics is largely confined to the normally matched base-pairs that are immediately adjacent to a mismatch/ unpaired base and could be used by MutS as a cue for mismatch-specific recognition. Interestingly, the enhanced dynamics associated with base-pairs adjacent to a mismatch are significantly restricted upon MutS binding, perhaps "resetting" the cues for downstream events that follow MutS binding. Recognition of such details of motional dynamics of DNA for the first time in the current study enabled us to propose a model that integrates the details of mismatch recognition by MutS as revealed by the high-resolution crystal structure with that of observed base dynamics, and unveils a minimal composite read-out involving the base mismatch and its adjacent normal base-pairs.

Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant.

No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for gammaE-crystallin. Recombinant protein for the mutant gammaE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of gammaE. Quantitative RT-PCR showed that gammaE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of gammaE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity.

A single mismatch in the DNA induces enhanced aggregation of MutS. Hydrodynamic analyses of the protein-DNA complexes.

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.