James C. Hu

The Gene Ontology's Reference Genome Project: A Unified Framework for Functional Annotation across Species

The Gene Ontology (GO) is a collaborative effort that provides structured vocabularies for annotating the molecular function, biological role, and cellular location of gene products in a highly systematic way and in a species-neutral manner with the aim of unifying the representation of gene function across different organisms. Each contributing member of the GO Consortium independently associates GO terms to gene products from the organism(s) they are annotating. Here we introduce the Reference Genome project, which brings together those independent efforts into a unified framework based on the evolutionary relationships between genes in these different organisms. The Reference Genome project has two primary goals: to increase the depth and breadth of annotations for genes in each of the organisms in the project, and to create data sets and tools that enable other genome annotation efforts to infer GO annotations for homologous genes in their organisms. In addition, the project has several important incidental benefits, such as increasing annotation consistency across genome databases, and providing important improvements to the GOs logical structure and biological content.

The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology

BackgroundSystems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion.ResultsWe have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway® entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics.ConclusionsThe E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.

Repressor fusions as a tool to study protein–protein interactions

Genetic reporter systems are necessarily limited in the kinds of information they can provide. The most obvious limitation is that the insides of cells are complex. The phenotype of a gene fusion protein will be determined not only by the property we wish to assay, but also by other effects of the intracellular environment on the chimeric gene product, including the following: protein folding in the cells used (usually E. coli or Saccharomyces cerevisiae), sensitivity to intracellular proteolysis, appropriate localization within the cell, and interactions with other cellular components. The strength of interactions needed to confer repressor activity is clearly dependent on the level of expression of the fusion protein. For repressor fusion proteins, our ability to detect assembled or weakly bound oligomers is limited by the fact that the N-terminal domain by itself will confer repressor activity at high expression levels. At the other extreme, for very tight dimers or tetramers only the most drastic mutations might lose enough activity to give a detectable change of phenotype.The significance of activity in the λ repressor system, or in similar genetic oligomerization reporters based on LexA repressor [4xConstruction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study. Schmidt-Dorr, T., Oertel, B.P., Pernelle, C., Bracco, L., Schnarr, M., and Granger, S.M. Biochemistry. 1991; 30: 9657–9664Crossref | PubMedSee all References][4], 434 repressor [15xDimerization of leucine zippers analyzed by random selection. Pu, W.T. and Struhl, K. Nucleic Acids Res. 1993; 21: 4348–4355Crossref | PubMed | Scopus (20)See all References][15], AraC protein [5xFunctional domains of the AraC protein. Bustos, S. and Schleif, R. Proc. Natl. Acad. Sci. USA. 1993; 90: 5638–5642Crossref | PubMedSee all References][5] and LuxR protein (DM Sitnikov, JC Hu and TO Baldwin, unpublished data) is necessarily limited. Genetic methods are not meant to be a substitute for rigorous biochemistry in determining oligomerization states and binding constants. That said, the ‘awesome power’ of genetics lies in the speed with which one can test the properties of astronomically large numbers of amino acid sequences. The need to test such numbers of sequences arises in searching for specific genes in large genomes, and our results suggest that dominant negative selections can be used to find novel partners for known dimers. Moreover, the ability to test large numbers of sequences has interesting implications for protein design. Fusion methods such as the ones described here will allow us to test populations of sequences that represent a family of guesses as to what is needed to form a desired structure. Imitating the evolution of naturally occurring proteins, we cannot only allow selection to identify the best candidates from such populations, but also allow additional rounds of mutagenesis and selection to refine their design.

Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate

BACKGROUNDnChemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands. However, few CID systems are currently available. The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system.nnnRESULTSnBisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR. Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed. Furthermore, the off-rate was found to be 0.0002 s(-1), approximately 100 times slower than that reported for DHFR-MTX. Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR(2) ternary complex.nnnCONCLUSIONSnThe combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo. Formation of the bisMTX:DHFR(2) ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex.

Genetic selection of short peptides that support protein oligomerization in vivo

An important goal in protein engineering is to control associations between designed proteins. This is most often done by fusing known, naturally occurring oligomerization modules, such as leucine zippers [1] [2] [3], to the proteins of interest [4] [5] [6]. It is of considerable interest to design or discover new oligomerization domains that have novel binding specificities [7] [8] [9] [10] [11] in order to expand the toolbox of the protein engineer and also to eliminate associations of the designed proteins with endogenous factors. We report here a simple genetic selection scheme through which to search libraries for peptides that are able to mediate homodimerization or higher-order self-oligomerization of a protein in vivo. We found several peptides that support oligomerization of the lambda repressor DNA-binding domain in Escherichia coli cells, some of them as efficiently as the endogenous dimerization domain or the GCN4 leucine zipper. Many are very small, comprising as few as six residues. This study strongly supports the notion that peptide sequence space is rich in small peptides, which might be useful in protein engineering and other applications.

Isolation and mapping of self-assembling protein domains encoded by the Saccharomyces cerevisiae genome using λ repressor fusions

Understanding how proteins are able to form stable complexes is of fundamental interest from the perspective of protein structure and function. Here we show that λ repressor fusions can be used to identify and characterize homotypic interaction domains encoded by the genome of Saccharomyces cerevisiae, using a selection for polypeptides that can drive the assembly of the DNA binding domain of bacteriophage λ repressor. Three high complexity libraries were constructed by cloning random fragments of S. cerevisiae DNA as λ repressor fusions. Repressor fusions encoding homotypic interactions were recovered, identifying oligomerization units in 35 yeast proteins. Seventeen of these interaction domains have not been previously reported, while the other 18 represent homotypic interactions that have been characterized at varying levels of detail. The novel interactions include several predicted coiled‐coils as well as domains of unknown structure. With the availability of genomic sequences it should be possible to apply this approach, which provides information about protein–protein interactions that is complementary to that obtained from yeast two‐hybrid screens, on a genome‐wide scale in yeast or other organisms where large‐scale protein–protein interaction data is not available. Copyright

Model systems: Studying molecular recognition using bacterial n-hybrid systems.

Since the first description of the yeast two-hybrid system, related genetic assays for protein-protein interactions have become popular and powerful tools for structure-function analysis on the scale of individual proteins or whole proteomes. After a somewhat surprising lag, similar systems have recently been described for use in bacterial hosts. n-hybrid modifications of the original yeast system have been used to examine interactions with DNA, RNA and small molecules, and other modifications have improved throughput for genomic applications. Bacterial n-hybrid systems are being designed for a similar array of uses. Will the bacterial systems be as popular as the yeast n-hybrid systems? Only time will tell.

Detection of tetramerization domains in vivo by cooperative DNA binding to tandem λ operator sites

Abstract Chimeric proteins comprising the N-terminal DNA binding domain of λ repressor fused to a fragment of a foreign protein have been used to detect oligomerization of the latter. Fusions containing dimeric and tetrameric leucine zipper domains can be distinguished based on their in vivo repressor activities on a pair of cat-lacZ reporter strains. Repressor fusions are unable to efficiently repress transcription from a synthetic promoter that overlaps a weak operator site; repression by tetrameric, but not dimeric, fusion proteins is increased by the presence of a strong, upstream operator site. To construct reporters we developed a shuttle system that allows rapid construction of single-copy operon fusions in E. coli , with both cat and lacZ as reporters.

The emerging world of wikis

We noted with interest the letter “Preserving accuracy in Gen Bank,” (M. I. Bidartondo et al. , 21 March, p. [1616][1]) and the related News of the Week story “Proposal to ‘wikify’ GenBank meets stiff resistance” (E. Pennisi, 21 March, p. [1598][2]). David Lipmans fears that wikifying

Specificity of the E. coli LysR-Type Transcriptional Regulators

Background Families of paralogous oligomeric proteins are common in biology. How the specificity of assembly evolves is a fundamental question of biology. The LysR-Type Transcriptional Regulators (LTTR) form perhaps the largest family of transcriptional regulators in bacteria. Because genomes often encode many LTTR family members, it is assumed that many distinct homooligomers are formed simultaneously in the same cell without interfering with each others activities, suggesting specificity in the interactions. However, this assumption has not been systematically tested. Methodology/Principal Findings A negative-dominant assay with λcI repressor fusions was used to evaluate the assembly of the LTTRs in E. coli K-12. Thioredoxin (Trx)-LTTR fusions were used to challenge the homooligomeric interactions of λcI-LTTR fusions. Eight cI-LTTR fusions were challenged with twenty-eight Trx fusions. LTTRs could be divided into three classes based on their interactions with other LTTRs. Conclusions/Significance Multimerization of LTTRs in E. coli K-12 is mostly specific. However, under the conditions of the assay, many LTTRs interact with more than one noncognate partner. The physiological significance and physical basis for these interactions are not known.