Lon Phan

dbSNP: the NCBI database of genetic variation

In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Center for Biotechnology Information (NCBI) has established the dbSNP database [S.T.Sherry, M.Ward and K. Sirotkin (1999) Genome Res., 9, 677-679]. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. The complete contents of dbSNP can also be downloaded in multiple formats via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/.

The NCBI dbGaP database of genotypes and phenotypes

The National Center for Biotechnology Information has created the dbGaP public repository for individual-level phenotype, exposure, genotype and sequence data and the associations between them. dbGaP assigns stable, unique identifiers to studies and subsets of information from those studies, including documents, individual phenotypic variables, tables of trait data, sets of genotype data, computed phenotype-genotype associations, and groups of study subjects who have given similar consents for use of their data.

Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2.

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre‐initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C‐termini of eIF5 and the catalytic (ϵ) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the β subunit of their common substrate eIF2. We also find that three lysine‐rich boxes in the N‐terminal segment of eIF2β mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bϵ employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine‐rich binding domain for these factors in their eIF2β homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1‐encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre‐initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

ABSTRACT Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in theSaccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

Development and characterization of a reconstituted yeast translation initiation system.

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo

Translation initiation factor 1A (eIF1A) is predicted to bind in the decoding site of the 40S ribosome and has been implicated in recruitment of the eIF2–GTP–Met‐tRNAiMet ternary complex (TC) and ribosomal scanning. We show that the unstructured C‐terminus of eIF1A interacts with the C‐terminus of eIF5B, a factor that stimulates 40S–60S subunit joining, and removal of this domain of eIF1A diminishes translation initiation in vivo. These findings support the idea that eIF1A–eIF5B association is instrumental in releasing eIF1A from the ribosome after subunit joining. A larger C‐terminal truncation that removes a 310 helix in eIF1A deregulates GCN4 translation in a manner suppressed by overexpressing TC, implicating eIF1A in TC binding to 40S ribosomes in vivo. The unstructured N‐terminus of eIF1A interacts with eIF2 and eIF3 and is required at low temperatures for a step following TC recruitment. We propose a modular organization for eIF1A wherein a core ribosome‐binding domain is flanked by flexible segments that mediate interactions with other factors involved in recruitment of TC and release of eIF1A at subunit joining.

Sequence variations in the public human genome data reflect a bottlenecked population history

Single-nucleotide polymorphisms (SNPs) constitute the great majority of variations in the human genome, and as heritable variable landmarks they are useful markers for disease mapping and resolving population structure. Redundant coverage in overlaps of large-insert genomic clones, sequenced as part of the Human Genome Project, comprises a quarter of the genome, and it is representative in terms of base compositional and functional sequence features. We mined these regions to produce 500,000 high-confidence SNP candidates as a uniform resource for describing nucleotide diversity and its regional variation within the genome. Distributions of marker density observed at different overlap length scales under a model of recombination and population size change show that the history of the population represented by the public genome sequence is one of collapse followed by a recent phase of mild size recovery. The inferred times of collapse and recovery are Upper Paleolithic, in agreement with archaeological evidence of the initial modern human colonization of Europe.

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation.

eIF5 stimulates the GTPase activity of eIF2 bound to Met‐tRNAiMet, and its C‐terminal domain (eIF5‐CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met‐tRNAiMet. The tif5‐7A mutation in eIF5‐CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met‐tRNAiMet and mRNA to 40S subunits in vitro. Interestingly, eIF5‐CTD bound simultaneously to the eIF4G subunit of the cap‐binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5‐7A eliminated eIF5 as a stable component of the pre‐initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate‐limiting defect in this mutant. We propose that eIF5‐CTD stimulates binding of Met‐tRNAiMet and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.

A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8‐PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co‐purified with this subcomplex, but not with distinct His8‐PRT1–TIF34–TIF35 (P45) or His8‐PRT1–TIF32 (P2) sub complexes. His8‐PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat‐treated prt1‐1 extracts are defective for Met‐tRNAiMet binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met‐ tRNAiMet and mRNA, and translation of luciferase mRNA, in a prt1‐1 extract almost as well with purified PN2 subcomplex as with five‐subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1–TIF32–NIP1 subcomplex.

Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding

eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met‐tRNAiMet and mRNA to the pre‐initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3–eIF5– eIF1–eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1–TIF34–TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome‐binding activity of eIF3.