Alejandro Merino

Functional organization of the yeast proteome by systematic analysis of protein complexes

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Oct-2 facilitates functional preinitiation complex assembly and is continuously required at the promoter for multiple rounds of transcription.

Octamer factor 2 (Oct‐2, OTF‐2, NF‐A2) is an ‘upstream’ promoter factor that binds to the octamer motif (ATGCAAAT) implicated in control of immunoglobulin gene transcription in B‐lymphocytes. We have studied the role of Oct‐2 in the process of transcription initiation in vitro using both nuclear extracts and purified basal transcription factors. Oct‐2 specifically stimulates transcription from octamer‐containing promoters in both systems. Thus, Oct‐2 is a ‘true activator’, rather than merely an ‘anti‐repressor’ counteracting the effect of histones. In order‐of‐addition experiments, Oct‐2 is required early, together with TFIID, to allow formation of a preinitiation complex. Oct‐2 cannot functionally interact with cloned TATA binding protein (TBP) but rather requires ‘coactivators’ found in the TFIID fraction. In single‐round transcription experiments, early competition for Oct‐2 by an octamer oligonucleotide is deleterious, but no effect is seen after assembly of a complete preinitiation complex. However, for multiple rounds of transcription, Oct‐2 is continuously required at the promoter; this result argues against a ‘hit‐and‐run’ mechanism whereby the activator becomes dispensible after organizing a TFIID‐promoter complex. In agreement with our previous studies in vivo, the N‐terminal glutamine‐rich activation domain of Oct‐2 is required for full activity in vitro, indicating that this domain directly interacts with basal transcription factors.