ABSTRACT
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.
Subject(s)
DNA Polymerase I/metabolism , Models, Molecular , Pol1 Transcription Initiation Complex Proteins/chemistry , Pol1 Transcription Initiation Complex Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cell Proliferation , Humans , Molecular Sequence Data , Mutation , Pol1 Transcription Initiation Complex Proteins/genetics , Promoter Regions, Genetic , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Serine/metabolismABSTRACT
Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking 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.
Subject(s)
Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Subunits/chemistry , RNA Polymerase III/chemistry , Saccharomyces cerevisiae/enzymology , Catalytic Domain , Cross-Linking Reagents/pharmacology , Light , Lysine/metabolism , Models, Molecular , Protein Binding/drug effects , Protein Binding/radiation effects , Protein Interaction Maps/drug effects , Protein Interaction Maps/radiation effects , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/metabolism , Protein Transport/drug effects , Protein Transport/radiation effects , RNA Polymerase III/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/radiation effects , Structure-Activity RelationshipABSTRACT
RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II-TFIIS-TFIIF-TFIIE complex.
Subject(s)
RNA Cleavage , RNA Polymerase I/chemistry , Cross-Linking Reagents , Lysine/chemistry , Mass Spectrometry , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA Polymerase I/metabolism , RNA, Ribosomal/metabolism , Transcriptional Elongation Factors/metabolismABSTRACT
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 A 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.