A comprehensive analysis of protein-protein interactions in ...

A comprehensive analysis of protein-protein interactions in ...

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae Article by Peter Uetz, et.al. Presented by Kerstin Obando Background Information In April 1996 the genome sequence of Saccharomyces cerevisiae, a budding yeast was completed.

Enabling an eukaryotic organism to be analyzed on a genomic scale for the first time. A problem with two different approaches to a solution Problem: What are the roles of the 6,000

gene products and how do they interact to create a eukaryotic organism? Solution: Two-hybrid analysis will produce additional information that can place yeast ORFs within a biological context. Two hybrid analysis Separates the coding sequences for the DNA-binding and activation domains of a transcriptional activator and clones them into separate vector molecules.

The coding sequence of a candidate protein whose partners are sought is then fused with the DNA-binding domain, this is known as the bait. A library of coding sequences for proteins that might interact with the bait, called the prey, is made in fusion with the activation domain. If the bait and prey physically interact, the DNA-binding and activation domains are closely juxtaposed and the reconstituted transcriptional activator can mediate the switching on of the gene that effects the color change. Basically They have two proteins that want to test to see if

they can physically associate. They attach each of them to separate fragments of a third protein, called a transcription activator, which has the ability to switch on genes. If the two proteins interact, then the two fragments of the activator are reunited and a gene is turned on that produces an easily monitored color change in the yeast cells. Protein array of activation-domain hybrids

An array of hybrid proteins was constructed to examine the protein activity in a format that allowed the assay of every predicted ORF. The hybrid array used in this study was a set of yeast colonies derived from about 6,000 individual transformants. The transformation event inserts one of the yeast ORFs into a Gal4 transcription-activation domain vector to create a hybrid protein. To enable rapid, large scale transformation, they generated the ORFs as a set of PCR products with 70bp sequences at their 5 and 3 ends that precisely matched sequences in

the activation domain vector pOAD. More on the protein array After transformation of a yeast two hybrid reporter strain , they pooled two colonies from each transformation plate to constitute a single array element with the entire array contained on 16 microassay plates of 384 colonies each. Strains expressing different bait molecules, 192 in all, were mated to each member of the array and the positive interactions were identified on His3

deficient plates. Results for this assay 87 of the 192 DNA-binding domain hybrids screened were identified in a putative protein-protein interaction, resulting in 281 interacting proteins. Exclusion of some known interactions occurred due to unknown variability within the data.

High-throughput screen of an activationdomain library As an alternative method this team developed highthroughput screens based on a library made by pooling transformants containing 6,000 potential ORFs fused to the Gal4 activation domain In contrast to the array, the library screening method does not keep cells expressing the 6,000 prey molecules separate. Instead it pools them and each of the 6,000 strains expressing a different bait is mated with the pool. Hybrid cells are selected and then screened for positive interactions

More on the Library The same PCR products and recipient plasmids were used to generate two collections of transformants, each consisting of 64 barcoded 96well plates. 87% were successfully cloned into both plasmids. After mating each DNA-binding domain to the activation domain, they were transferred to selective plates to select interacting pairs that activated URA3 and lacZ reporter genes.

Results for the library 817 yeast ORFs (15%) were identified in a putative protein-protein interaction, resulting in 692 interacting protein pairs. Interactions identified Independent experiment 286 (41%) Multiple times in single exp. 186 (27%) Only once 220 (32%)

Comparison of the approaches The two strategies yield different results. The array method is more efficient, which is only partly attributable to the judicious choice of baits. The library approach, while benefiting from much high throughput, has the disadvantage that cells in the prey pool compete with one another during mating, so selecting against cell expressing fusion products that retard either process. Thus, of the 12 baits that gave positive interactions with both screens 48 possible partners were identified by the array approach, against only 14 in the library screen.

Visual Comparison Top: the array of haploid transfomants on leucine minus plates. Bottom: Diploid colonies after two weeks of growth on a tryptophan minus plate.

Discussion Arrays of biomolecules possess unique advantages for the handling and investigation of multiple samples. They provide a fixed location for each element such that those scoring positive in an assay are immediately identified; they have the capacity to be comprehensive and of high density. The high throughput library approach is reasonable to employ in order to complete a screen of all encoded ORFs of an organism, however the array approach, while more time and labor intensive would probably provide more

positives. Three types of Discoveries 1) Guilty by association - Interactions between proteins of known and unknown function have indeed allowed the role of the latter to be inferred. 2) Unrecognized interactions have been identified between proteins involved in the same biological process. 3) The screen has provided clues for seeing how

individual biological events are integrated into larger cellular process. Quick Overview This study was interested in examining the putative protein-protein interactions identified in these screens in reference to there functional roles according to the yeast protein database. 32% of the interactions found were between proteins with no functional classification. This observation indicates that there may be a

significant number of as yet undiscovered pathways and/or complexes that can be identified using systematic approaches. Illustration of Interaction An example of the data analysis software that illustrates the interactions revealed and provides an overview of the

mechanisms involved. Questions?

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