TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more 'metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei

RIBFIND2: identifying rigid bodies in protein and nucleic acid structures

Molecular structures are often fitted into cryo-EM maps by flexible fitting. When this requires large conformational changes, identifying rigid bodies can help optimize the model-map fit. Tools for identifying rigid bodies in protein structures exist, however an equivalent for nucleic acid structures is lacking. With the increase in cryo-EM maps containing RNA and progress in RNA structure prediction, there is a need for such tools. We previously developed RIBFIND, a program for clustering protein secondary structures into rigid bodies. In RIBFIND2, this approach is extended to nucleic acid structures. RIBFIND2 can identify biologically relevant rigid bodies in important groups of complex RNA structures, capturing a wide range of dynamics, including large rigid-body movements. The usefulness of RIBFIND2-assigned rigid bodies in cryo-EM model refinement was demonstrated on three examples, with two conformations each: Group II Intron complexed IEP, Internal Ribosome Entry Site and the Processome, using cryo-EM maps at 2.7–5 Å resolution. A hierarchical refinement approach, performed on progressively smaller sets of RIBFIND2 rigid bodies, was clearly shown to have an advantage over classical all-atom refinement. RIBFIND2 is available via a web server with structure visualization and as a standalone tool

Dissecting the role of the Ribosome in Cotranslational Folding using CRISPR-Cas9

The ribosome is the universal orchestrator of protein synthesis and in vivo many proteins begin to fold cotranslationally while they are being synthesised on the ribosome. Emerging evidence suggests that the ribosome plays an active role to influence the folding pathway and prevent misfolding. Burrowing through the exit tunnel to the ribosome surface, the nascent chain experiences a unique environment. Here we apply CRISPR-Cas9 genome editing to dissect the influence of the ribosomal exit tunnel proteins on cotranslational folding. The ribosomal protein loops of uL4, uL22, uL23 and uL24 contribute to the geometry of the tunnel and are highly conserved yet display some interesting evolutionary differences across the three domains of life. These protein loops necessarily interact with the diverse range of nascent polypeptides in the proteome. We rationally designed and generated a series of ribosomal variants with altered tunnel geometries via truncations and insertions in ribosomal protein tunnel loops. An integrated approach was used to characterise ribosomal variants combining biochemical assays, NMR spectroscopy, cryo-EM, and molecular dynamics simulations. Cotranslational folding of an immunoglobulin-like domain, the filamin FLN5, can be both enhanced or inhibited with different ribosomal loop variants, with the loops of uL23 and uL24 in the vestibule having a pronounced effect. In depth investigation of the mechanistic basis for earlier folding of these ribosomal variants is presented, allowing us to define the modulation of the free energy landscape for folding. This study is the first systematic application of CRISPR-Cas9 genome editing to the ribosomal exit tunnel and thus contributes to understanding key questions regarding the earliest encounters of newly synthesising polypeptides, specifically how the geometry of the exit tunnel influences the folding of the nascent chain and modulates its folding pathway

Modulating co-translational protein folding by rational design and ribosome engineering

The narrow exit tunnel of the ribosome is important for cotranslational protein folding. Here, authors show that their rationally designed and engineered exit tunnel protein loops modulate the free energy of nascent chain dynamics and folding