From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

A key to defining the function of any protein is to determine what it binds to. In the case of protein-protein interactions, the association of a protein of unknown function with one whose function is known can provide a useful and compelling “guilt by association.” The techniques used in this effort are quite varied.

Comparisons of Genome Composition Although not evidence of direct association, the mere presence of combinations of genes in particular genomes can hint at

FIGURE 9–22 DNA microarray. A microarray can be prepared from any known DNA sequence, from any source, generated by chemical synthesis or by PCR. The DNA is positioned on a solid surface (usually specially treated glass slides) with the aid of a robotic device capable of depositing very small (nanoliter) drops in precise patterns. UV light cross-links the DNA to the glass slides. Once the DNA is at tached to the surface, the microarray can be probed with other fluorescently labeled nucleic acids. Here, mRNA samples are collected from cells at two different stages in the development of a frog. The cDNA probes are made with nucleotides that fluoresce in different colors for each sample; a mixture of the cDNAs is used to probe the microarray. Green spots represent mRNAs more abundant at the single-cell stage; red spots, sequences more abundant later in devel opment. The yellow spots indicate approximately equal abundance at both stages.

protein function. We can simply search the genomic databases for particular genes, then determine what other genes are present in the same genomes (Fig. 9–24). When two genes always appear together in a genome, it suggests that the proteins they encode may be functionally related. Such correlations are most useful if the function of at least one of the proteins is known.

Purification of Protein Complexes With the construction of cDNA libraries in which each gene is contiguous with (fused to) an epitope tag, workers can immunoprecipitate the protein product of a gene by using the antibody that binds to the epitope (Fig. 9–15b). If the tagged protein is expressed in cells, other proteins that bind to it may also be precipitated with it. Identification of the associated proteins reveals some of the protein-protein interactions of the tagged protein. There are many variations of this process. For example, a crude extract of cells that express a similarly tagged protein is added to a column containing immobilized antibody. The tagged protein binds to the antibody, and proteins that inter act with the tagged protein are sometimes also retained on the column. The connection between the protein and the tag is cleaved with a specific protease, and the protein complexes are eluted from the column and analyzed. Researchers can use these methods to define complex networks of interactions within a cell. A variety of useful protein tags are available. A common one is a histidine tag, often just a string of six His residues. A poly-His sequence binds quite tightly to metals such as nickel. If a protein is cloned so that its sequence is contiguous with a His tag, it will have the extra His residues at its carboxyl terminus. The protein can then be purified by chromatography on columns with immobilized nickel. These procedures are convenient but require caution, because the additional amino acid residues in an epitope or His tag can affect protein activity.

FIGURE 9–23 Enlarged image of a DNA microarray. Each glowing spot in this microarray contains DNA from one of the 6,200 genes of the yeast (S. cerevisiae) genome, with every gene represented in the array. The microarray has been probed with fluorescently labeled nucleic acid derived from the mRNAs obtained (1) when the cells were growing normally in culture and (2) five hours after the cells began to form spores. The green spots represent genes expressed at higher levels during normal growth; the red spots, genes expressed at higher levels during sporulation. The yellow spots represent genes that do not change their levels of expression during sporulation. This image is enlarged; the microarray actually measures only 1.8X1.8 cm.

FIGURE 9–24 Use of comparative genomics to identify functionally related genes. One use of comparative genomics is to prepare phylogenetic profiles in order to identify genes that always appear together in a genome. This example shows a comparison of genes from four organisms, but in practice, computer searches can look at dozens of species. The designations P1, P2, and so forth refer to proteins en coded by each species. This technique does not require homologous proteins. In this example, because proteins P3 and P6 always appear together in a genome they may be functionally related. Further testing would be needed to confirm this inference.

Yeast Two-Hybrid Analysis A sophisticated genetic approach to defining protein-protein interactions is based on the properties of the Gal4 protein (Gal4p), which activates transcription of certain genes in yeast (see Fig. 28–28). Gal4p has two domains, one that binds to a specific DNA sequence and another that activates the RNA polymerase that synthesizes mRNA from an adjacent reporter gene. The domains are stable when separated, but activation of the RNA polymerase requires interaction with the activation domain, which in turn requires positioning by the DNA-binding domain. Hence, the do mains must be brought together to function correctly (Fig. 9–25a).

FIGURE 9–25 The yeast two-hybrid system. (a) In this system for de tecting protein-protein interactions, the aim is to bring together the DNA-binding domain and the activation domain of the yeast Gal4 protein through the interaction of two proteins, X and Y, to which each domain is fused. This interaction is accompanied by the expression of a reporter gene. (b) The two fusions are created in separate yeast strains, which are then mated. The mated mixture is plated on a medium on which the yeast cannot survive unless the reporter gene is expressed. Thus, all surviving colonies have interacting protein fusion pairs. Sequencing of the fusion proteins in the survivors reveals which proteins are interacting.

In this method, the protein-coding regions of genes to be analyzed are fused to the coding sequences of ei ther the DNA-binding domain or the activation domain of Gal4p, and the resulting genes express a series of fusion proteins. If a protein fused to the DNA-binding domain interacts with a protein fused to the activation domain, transcription is activated. The reporter gene transcribed by this activation is generally one that yields a protein required for growth, or is an enzyme that catalyzes a reaction with a colored product. Thus, when grown on the proper medium, cells that contain such a pair of interacting proteins are easily distinguished from those that do not. Typically, many genes are fused to the Gal4p DNA-binding domain gene in one yeast strain, and many other genes are fused to the Gal4p activation domain gene in another yeast strain, then the yeast strains are mated and individual diploid cells grown into colonies (Fig. 9–25b). This allows for large-scale screening for proteins that interact in the cell.

All these techniques provide important clues to pro tein function. However, they do not replace classical biochemistry. They simply provide researchers with an expedited entrée into important new biological problems. In the end, a detailed functional understanding of any new protein requires traditional biochemical analyses—such as were used for the many well-studied proteins described in this text. When paired with the simultaneously evolving tools of biochemistry and molecular biology, genomics and proteomics are speeding the discovery not only of new proteins but of new biological processes and mechanisms.

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قسم الشؤون الفكرية وجامعة ميسان يبحثان تعزيز التعاون العلمي المشترك

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مصر تحقق إنجازاً دولياً جديداً في منظمة الصحة العالمية

دراسة: السباحة فعّالة أكثر من الجري في الحفاظ على صحة القلب

عامل خطر في بداية الحمل يؤخر تطور الكلام والمهارات الحركية لدى الرضع

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مواضيع ذات صلة

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins

DNA Cloning: The Basics:- Expression of Cloned Genes Produces Large Quantities of Protein

DNA Cloning: The Basics:- Specific DNA Sequences Are Detectable by Hybridization

DNA Cloning: The Basics:- Cloning Vectors Allow Amplification of Inserted DNA Segments

DNA Cloning: The Basics:- Restriction Endonucleases and DNA Ligase Yield Recombinant DNA

Other Functions of Nucleotides:- Adenine Nucleotides Are Components of Many Enzymes Cofactors

Other Functions of Nucleotides:- Nucleotides Carry Chemical Energy in Cells