RNA polymerase III subunit architecture and implications for open promoter complex formation

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine crosslinking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization

An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.Peer reviewe

Role of serum brain derived neurotrophic factor and central N-acetylasparate for clinical response under antidepressive pharmacotherapy

Background: The predictive therapeutic value of brain derived neurotrophic factor (BDNF) and its changes associated with the use of specific antidepressants are still unclear. In this study, we examined BDNF as a peripheral and NAA as a central biomarker over the time course of antidepressant treatment to specify both of their roles in the response to the medication and clinical outcome. Methods: We examined serum BDNF (ELISA kit) in a sample of 76 (47 female and 29 male) depressed patients in a naturalistic setting. BDNF was assessed before medication and subsequently after two, four and six weeks of antidepressant treatment. Additionally, in fifteen patients, N-acetylaspartate (NAA) was measured in the anterior cingulate cortex (ACC) with magnetic resonance spectroscopy (MRS). Over a time course of six weeks BDNF and NAA were also examined in a group of 41 healthy controls. Results: We found significant lower serum BDNF concentrations in depressed patients compared to the sample of healthy volunteers before and after medication. BDNF and clinical symptoms decreased significantly in the patients over the time course of antidepressant treatment. Serum BDNF levels at baseline predicted the symptom outcome after eight weeks. Specifically, responders and remitters had lower serum BDNF at baseline than the nonresponders and nonremitters. NAA was slightly decreased but not significantly lower in depressed patients when compared with healthy controls. During treatment period, NAA showed a tendency to increase. Limitations: A relative high drop-out rate and possibly, a suboptimal observation period for BDNF. Conclusion: Our data confirm serum BDNF as a biomarker of depression with a possible role in response prediction. However, our findings argue against serum BDNF increase being a prerequisite to depressive symptom reduction

Functional architecture of RNA polymerase I

Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 Å cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3′-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3′-end trimming

Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth

Cell growth is regulated during RNA polymerase (Pol) I transcription initiation by the conserved factor Rrn3/TIF-IA in yeast/humans. Here we provide a structure-function analysis of Rrn3 based on a combination of structural biology with in vivo and in vitro functional assays. The Rrn3 crystal structure reveals a unique HEAT repeat fold and a surface serine patch. Phosphorylation of this patch represses human Pol I transcription, and a phospho-mimetic patch mutation prevents Rrn3 binding to Pol I in vitro and reduces cell growth and Pol I gene occupancy in vivo. Cross-linking indicates that Rrn3 binds Pol I between its subcomplexes, AC40/19 and A14/43, which faces the serine patch. The corresponding region of Pol II binds the Mediator head that cooperates with transcription factor (TF) IIB. Consistent with this, the Rrn3-binding factor Rrn7 is predicted to be a TFIIB homolog. This reveals the molecular basis of Rrn3-regulated Pol I initiation and cell growth, and indicates a general architecture of eukaryotic transcription initiation complexes

Structure of eukaryotic RNA polymerases.

The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions