ABSTRACT
Multi-subunit RNA polymerases (RNAPs) in all three domains of life share a common ancestry. The composition of the archaeal RNAP (aRNAP) is not identical between phyla and species, with subunits Rpo8 and Rpo13 found in restricted subsets of archaea. While Rpo8 has an ortholog, Rpb8, in the nuclear eukaryal RNAPs, Rpo13 lacks clear eukaryal orthologs. Here, we report crystal structures of the DNA-bound and free form of the aRNAP from Sulfolobus shibatae. Together with biochemical and biophysical analyses, these data show that Rpo13 C-terminus binds non-specifically to double-stranded DNA. These interactions map on our RNAP-DNA binary complex on the downstream DNA at the far end of the DNA entry channel. Our findings thus support Rpo13 as a RNAP-DNA stabilization factor, a role reminiscent of eukaryotic general transcriptional factors. The data further yield insight into the mechanisms and evolution of RNAP-DNA interaction.
Subject(s)
Archaeal Proteins/chemistry , DNA-Directed RNA Polymerases/chemistry , DNA/chemistry , Apoproteins/chemistry , Archaeal Proteins/metabolism , Crystallography, X-Ray , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Models, Molecular , Protein Subunits/chemistry , Protein Subunits/metabolism , Sulfolobus/enzymologyABSTRACT
We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae. We compare the three crystal forms in which this RNAP packs (space groups P212121, P21212 and P21) and provide a preliminary biophysical characterization of the newly identified 13-subunit Rpo13. The availability of different crystal forms for this RNAP allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. We observe the pivotal role played by the protruding stalk composed of subunits Rpo4 and Rpo7 in the lattice contacts. Aided by MALLS (multi-angle laser light scattering), we have initiated the biophysical characterization of the recombinantly expressed and purified subunit Rpo13, a necessary step towards the understanding of Rpo13's role in archaeal transcription.
Subject(s)
Archaeal Proteins/chemistry , DNA-Directed RNA Polymerases/chemistry , Protein Subunits/chemistry , RNA, Archaeal/genetics , Sulfolobus/enzymology , Sulfolobus/genetics , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Subunits/genetics , Protein Subunits/metabolism , Transcription, GeneticABSTRACT
Recent work has identified a "glutamate switch" in six of the seven clades of AAA+ ATPases. The glutamate switch acts to transduce information regarding substrate binding to the ATPase active site. We provide biochemical evidence that a highly conserved threonine residue acts as a glutamate switch in the replicative helicase, MCM, and, thus, reveal that the glutamate switch is a feature common to all seven AAA+ clades.
