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The geometric relationship between the two protein degradation signals of proteasome substrates, the polyubiquitin chain and the unfolded region, is dictated by the locations of their recognition sites on the proteasome. The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated degradation.

The proteasome initiates protein degradation at disordered regions within substrates. The proteasomal sequence preferences for the amino acid composition of these regions identified here affect protein half-life and explain unusual stability trends. The proteasome controls the concentrations of most proteins in eukaryotic cells. It recognizes its protein substrates through ubiquitin tags and initiates degradation at disordered regions within the substrate. Here we show that the proteasome has pronounced preferences for the amino acid sequence of the regions at which it initiates degradation. Specifically, proteins in which the initiation regions have biased amino acid compositions show longer half-lives in yeast than proteins with unbiased sequences in the regions. The relationship is also observed on a genomic scale in mouse cells. These preferences affect the degradation rates of proteins in vitro , can explain the unexpected stability of natural proteins in yeast and may affect the accumulation of toxic proteins in disease. We propose that the proteasome's sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis 1 , 2 . The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors 2 , 3 . Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans , which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules. Double-strand DNA break repair by the non-homologous end joining pathway involves the transition from a complex that bridges the DNA ends to a complex that aligns the DNA for ligation through the dissociation of the kinase subunits of the DNA-PK complexes.

Rad23 is an adaptor protein that binds to both ubiquitinated substrates and to the proteasome. Despite its association with the proteasome, Rad23 escapes degradation. Here we show that Rad23 remains stable because it lacks an effective initiation region at which the proteasome can engage the protein and unfold it. Rad23 contains several internal, unstructured loops, but these are too short to act as initiation regions. Experiments with model proteins show that internal loops must be surprisingly long to engage the proteasome and support degradation. These length requirements are not specific to Rad23 and reflect a general property of the proteasome. Rad23 accompanies ubiquitinated substrates to the proteasome for destruction but manages to avoid degradation. In this study, Fishbain et al. show that Rad23 escapes because it lacks an effective initiation region; therefore, the proteasome is unable to engage the protein and unfold it.

RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.

If the diversification of microbial life can be depicted as a single tree, as inferred by comparative sequencing of ribosomal RNAs, this could provide a framework for defining the order of emergence of new metabolic pathways. However, recent recognition that lateral gene transfer has been a significant force in microbial evolution has created uncertainty about the interpretation of taxonomies based on gene sequences. In this context, the origins and evolution of sulfate respiration will be evaluated considering the evolutionary history of a central enzyme in this process, the dissimilatory sulfite reductase. These studies suggest at least two major lateral transfer events during the early diversification of sulfate respiring microorganisms. The high sequence conservation of this enzyme has also provided a mechanism to directly explore the natural diversity of sulfate-respiring organisms using molecular techniques, avoiding the bias of culture-based identification. These studies suggest that the habitat range and evolutionary diversity of this key functional group of organisms is greater than now appreciated.

RNA Polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here we use cryo-electron microscopy to visualize the complex of TFIIIA and TFIIIC bound to the promoter. Brf1-TBP binding further stabilizes the DNA, resulting in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the mechanism of how the transcription initiation complex assembles on the 5S rRNA promoter, a crucial step in Pol III transcription regulation.

An obligately methylotrophic organism was isolated from a water well that manifested symptoms of biofouling. The isolate was appendaged and utilized methylamine, dimethylamine, trimethylamine, or methanol as the sole carbon and energy source. The isolate exhibited hydroxypyruvate reductase activity, suggesting C1-assimilation via the serine pathway. Fatty acid profiling indicated the predominance of 18:1 cis-fatty acids. The isolate did not grow anaerobically with nitrate as the final electron acceptor. Genomic DNA from the isolate did not hybridize against the narG gene, which encodes the alpha subunit of dissimilatory nitrate reductase in Escherichia coli. The phenotypic data suggested the assignment of the isolate to the genus Hyphomicrobium. The identification was supported by phylogenetic characterization based on 16S rRNA sequence comparisons of the isolate.