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Screening for suppressor mutations, i.e. mutations in one gene that partially or fully compensate for a mutation in another. Have been widely employed in Drosophila and yeast
Limitations : One potential problem is that the suppressor mutation may map to the same gene as the primary mutation, since second mutations in the same gene can suppress the primary mutant phenotype by introducing a compensatory conformational change within the same protein Even if the suppressor maps to a different gene, the two gene products might not actually interact. For example, a mutation that abolishes the activity of an enzyme required for amino acid biosynthesis could be suppressed by a gain of function mutation in a transport protein that increases the uptake of that amino acid from the environment. Furthermore, the mutations do not necessarily have to change the structures of proteins X and Y.
Enhancer mutations, i.e. those that worsen the phenotype generated by a primary mutation
One example of this strategy is the synthetic lethal screen, where individual mutations in the genes for proteins X and Y do not prevent interaction and are therefore viable, but simultaneous mutations in both genes prevent the interaction and result in a lethal phenotype
Investigators established a synthetic genetic array (SGA) system in which a mutation in one yeast gene can be crossed to a set of 5000 viable deletion mutants, allowing synthetic interactions to be mapped in a systematic fashion. This can be used to identify all the proteins involved in the same pathway or complex as a particular query protein Mutations in different genes that generate similar phenotypes often indicate that the protein products are part of the same complex or the same biochemical or signaling pathway For pathways, the order of protein function can often be established by epistasis. In this type of experiment, loss-offunction and gain-of-function mutations (with opposite phenotypes) are combined in the same cell or organism. If a loss-of-function mutation in gene X overrides a gain-offunction mutation in gene Y, it suggests that protein X acts downstream of protein Y in the pathway.
Co - immunoprecipitation
Affinity Chromatography
Interacting proteins will be retained on the column as a conjoined complex
Energy is transferred from an excited donor fluorophore to a nearby acceptor fluorophore. FRET occurs only when the two fluorophores are up to 10 nm apart, and can be detected by the change in the emission wavelength of the acceptor fluorophore.
Traditional protein interaction analysisTechniques s.a co-immunoprecipitation, affinity chromatography,and cross-linking used previously to characterize the interactions of individual proteins. X-ray crystallography and NMR spectroscopy that can be used to characterize protein interactions at the atomic level. These methods cant be used for high throughput analysis , hence development of functional genomics techniques.
The aim of functional genomics is to determine functions for the many anonymous genes and cDNAs amassing in databases, highthroughput strategies that link genes and proteins into functional networks are essential.
Standard cDNA expression libraries Phage display method The yeast two-hybrid system
M13 (a filamentous phage containing ss-DNA encased in a protein coat): contains five coat proteins, two of which are gVIIIp (gene VIII protein) and gIIIp (gene III protein).
matrix approach
Traditional clone-based library not an ideal platform for proteomewide interaction screening (Drawbacks-labor-intensive and technically demanding screening procedures). Phage display is a more suitable alternative the expression of fusion proteins in such a way that a foreign peptide sequence is displayed on the bacteriophage surface. Libraries of phage can be produced and screened to identify peptides that interact with a given probe (such as an antibody) which is immobilized on a membrane or in the well of a microtiter plate. Screening is basically a reiterative affinity-purification process, in which non-interacting phages are discarded and bound phages are eluted and used to reinfect E. coli. After several rounds of such panning the remaining, tightly-bound phage are isolated and the inserts sequenced to identify the interacting peptides
The major disadvantage of all in vitro library systems is that interactions occur in an unnatural environment where the protein may be incorrectly folded or partially unfolded.
Is the prototype of a range of related techniques in which protein interactions are assayed in vivo. The principle of the system is that proteins often comprise several functionally independent domains, which can function not only when they are covalently linked in the same polypeptide chain, but also when they are brought together through noncovalent interactions.
Transcription factors generally contain independent DNA-binding and transactivation domains, and this means that a functional transcription factor can be created if separately expressed DNA-binding and transactivation domains can be persuaded to interact. The general strategy is as follows: protein X is expressed as a fusion (a hybrid) with the DNA-binding domain of a transcription factor to generate a bait. A library of prey is then generated in which each clone is expressed as a fusion protein with the transcription factors transactivation domain.