To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the elongation complex in a new state, which was refined at 3.4-A resolution and identified as a possible translocation intermediate. The DNA base entering the active site occupies a 'pretemplating' position above the central bridge helix, which is shifted and occludes the templating position. A leucine residue in the trigger loop forms a wedge at the shifted bridge helix, but moves by 13 A to close the active site during nucleotide incorporation. Our results support a Brownian ratchet mechanism that involves swinging of the trigger loop between open, wedged and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.
G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.
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.
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