Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Abstract

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.mass spectrometry ͉ nascent RNP ͉ RNA processing ͉ transcription ͉ affinity purification P urification and mass spectrometric analysis of protein complexes is a ubiquitous approach in modern molecular biology (1). The identification of interacting proteins and their subsequent characterization provides valuable insight into the complexes that carry out essential cellular functions. Purifications can use multiple biochemical fractionations (e.g., ion exchange, gradients, gel filtration, and affinity purification) or a streamlined tandem affinity purification (TAP) approach (2). Conventional purification methods, however, often preclude the accurate identification of in vivo-relevant complexes. Major complications include dissociation of protein complexes, as a function of off-rates and extract dilution. Moreover, strong affinities can promote in vitro associations that do not exist in vivo. These can occur as a consequence of protein exchange and the absence of subcellular localization. Finally, major complexes may be insoluble, or harsh methods required to effect solubility can perturb complex composition. Ribonucleoprotein particles (RNPs) and especially transcription complexes are particularly subject to these caveats.The synthesis of protein-encoding mRNAs is directed by RNA polymerase II (pol II), an extensively studied macromolecular machine. The composition of pol II is dynamic and changes with the stage of transcription, namely, initiation, elongation, and termination (3, 4). Difficulties with complex purification underlie controversies, for example, to what extent initiation occurs by stepwise assembly of a preinitiation complex on DNA. Moreover, elongating pol II is tightly associated with DNA, which makes its characterization challenging (5).ChIP is a major tool for characterizing DNA-associated proteins and complexes, including transcription and cotranscriptional ...

Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis

“…This result reaffirms that the QTAX strategy is effective in capturing both stable and/or weak/transient protein interactions. A similar observation was reported for RNA polymerase II-interacting proteins when a similar strategy was applied for capturing its interactors by in vivo formaldehyde cross-linking and TAP (18).…”

Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

“…The interactions of the S. cerevisiae protein Npl3, with Air2, Ded1, Gbp2, Snp1, and Yra1 were reported in large-scale studies elsewhere, detected by AP-MS and/or yeast two-hybrid techniques (16,(27)(28)(29). As such, it was important for these to be first verified in the bacterial C2H system before the effect of arginine methylation was examined.…”

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…To circumvent these problems, in vivo chemical crosslinking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10 -16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12)(13)(14)(15)(16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues.…”