A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

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Gene expression from plasmids containing the araBAD promoter can be regulated by the concentration of arabinose in the growth medium. Guzman et al. The ability to express a cloned gene under controlled conditions is often very useful. In Escherichia coli, plasmid-based inducible promoter systems have been implemented using bacterial, phage, and chimeric promoters. These systems have been generally designed to respond to an external inducer by expressing high levels of the gene product(s) of interest, often for the purpose of obtaining material for purification. Plasmid systems also have been designed to have low basal levels of expression to minimize the effects of exposing cells to toxic gene products during growth.Plasmid systems based on the lac promoter are notoriously leaky; repression is often incomplete due to a combination of plasmid copy number effects and the absence of secondary operators required for the full range of gene control in the natural lac operon (1). Background expression levels should be lower in systems based on positive rather than negative control. Recently, expression plasmids based on the araBAD promoter (P araBAD ) have been constructed by Guzman et al. (2). Because the ara system can be induced by arabinose and is repressed by both catabolite repression in the presence of glucose or by competitive binding of the anti-inducer fucose, these plasmids have very low background levels of expression. In addition, gene expression can be turned on and off rapidly by changing the sugars in the medium.In addition to providing material for biochemical studies, the ability to conditionally control the expression of specific genes is useful for understanding how the presence or absence of the genes of interest affects the physiology of E. coli. Conditional expression also allows for selections and screens for mutations in other genes. For example, cells that express an essential gene under control of the araBAD promoter can be grown in the presence of arabinose, and then plated for growth on glucose to select for mutations that bypass the requirement for that function (2). Similarly, mutants that affect the toxicity of an expressed gene product could be isolated by selecting for growth in the presence of the inducer.For physiological and genetic studies, the very high levels of protein expressed by most inducible systems are often inappropriate. Ideally, one would like to be able to modulate gene expression over a range of levels. Guzman et al. (2) presented evidence that the pBAD vectors are also suitable for this purpose. Using alkaline phosphatase as a reporter, they showed that the levels of alkaline phosphatase in cultures grown in different concentrations of arabinose could be varied over an approximately 300-fold range. Moreover, expression could be set at intermediate levels by using inducer concentrations between 1.33 M and 133 M.

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 GO's logical structure and biological content.

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In the modern genomic era, scientists without extensive bioinformatic training need to apply high-power computational analyses to critical tasks like phage genome annotation. At the Center for Phage Technology (CPT), we developed a suite of phage-oriented tools housed in open, user-friendly web-based interfaces. A Galaxy platform conducts computationally intensive analyses and Apollo, a collaborative genome annotation editor, visualizes the results of these analyses. The collection includes open source applications such as the BLAST+ suite, InterProScan, and several gene callers, as well as unique tools developed at the CPT that allow maximum user flexibility. We describe in detail programs for finding Shine-Dalgarno sequences, resources used for confident identification of lysis genes such as spanins, and methods used for identifying interrupted genes that contain frameshifts or introns. At the CPT, genome annotation is separated into two robust segments that are facilitated through the automated execution of many tools chained together in an operation called a workflow. First, the structural annotation workflow results in gene and other feature calls. This is followed by a functional annotation workflow that combines sequence comparisons and conserved domain searching, which is contextualized to allow integrated evidence assessment in functional prediction. Finally, we describe a workflow used for comparative genomics. Using this multipurpose platform enables researchers to easily and accurately annotate an entire phage genome. The portal can be accessed at https://cpt.tamu.edu/ galaxy-pub with accompanying user training material.

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo-and heterodimers through a short ␣-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the (abcdefg) n heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C͞EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.The stoichiometry and specificity with which proteins interact is a key control point in many biological processes. For example, common dimerization domains allow transcription factors in the bZIP or bHLH-LZ families to form a variety of homo-and heterodimers with different properties. By expressing different sets of subunits under different conditions, cells can generate complex regulatory circuits from a relatively small number of genes. The correct functioning of this complex regulatory machinery depends on each of the component proteins assembling only with specific partners.Leucine zippers are an excellent model system to study how the stability and specificity of protein-protein interactions are determined. High-resolution x-ray crystallographic and NMR structures are available for several leucine zippers (1-7). As ␣-helical coiled coils, leucine zippers have simple secondary and tertiary structures. Moreover, the large number of naturally occurring leucine zipper proteins includes a wide variety of distinct and overlapping dimerization specificities. At the amino acid sequence level, leucine zippers are characterized by leucine appearing in every seventh position (d) over 4 to 5 heptad repeats (abcdefg) n . The hydrophobic core of the dimer interface is formed by residues at the a and d positions (Fig. 1); the solvent-accessible e and g positions are frequently occupied by charged amino acids (8, 9). In the crystal structures of leucine zippers, including GCN4 homodimers and Jun-Fos heterodimers, intersubunit salt bridges are seen between oppositely charged amino acids at the g (ith heptad) and eЈ (i ϩ 1th heptad of the other monomer)...

The Gene Ontology (GO) project is the largest resource for cataloguing gene function. The combination of solid conceptual underpinnings and a practical set of features have made the GO a widely adopted resource in the research community and an essential resource for data analysis. In this chapter, we provide a concise primer for all users of the GO. We briefly introduce the structure of the ontology and explain how to interpret annotations associated with the GO.