Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs’ structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

Detection of specific antibodies has numerous research, therapeutic and diagnostic applications. Short peptide ligands that bind specifically to antibodies with continuous epitopes can be derived from epitope mapping experiments. Short peptide ligands (mimotopes) specific to antibodies with discontinuous epitopes can be identified by screening complex peptide libraries. In an effort to enhance practical utility of such peptide ligands, we describe here a simple approach to turn such target antibody-specific peptide ligands into specific ELISA detection reagents. We show that a simple addition of biotinylated peptide ligands to commonly available horseradish peroxidase (HRP)-labeled streptavidin (or HRP-anti-biotin antibody), or digoxigenin-labeled peptides to HRP-anti-digoxigenin antibody detection reagents transformed these generic detection reagents into sensitive target antibody-specific reagents. ELISA assays performed using these reagents exhibited excellent analytical properties indicating their practical utility for antibody detection. One generic detection reagent can be readily transformed into many different specific ELISA reagents by a simple mix and match design using an appropriate target-specific peptide ligand. Simplicity of preparation of these ELISA reagents for detecting antibodies should facilitate their practical applications.

Promoter escape involves breaking of the favourable contacts between RNA polymerase (RNAP) and the promoter to allow transition to an elongation complex. The sequence of DNA template that is transcribed during promoter escape (ITS; Initially Transcribed Sequence) can affect promoter escape by mechanisms that are not yet fully understood. We employed a highly parallel strategy utilizing Next Generation Sequencing (NGS) to collect data on escape properties of thousands of ITS variants. We show that ITS controls promoter escape through a combination of position-dependent effects (most prominently, sequence-directed RNAP pausing), and position-independent effects derived from sequence encoded physical properties of the template (for example, RNA/DNA duplex stability). ITS often functions as an independent unit affecting escape in the same manner regardless of the promoter from which transcription initiates. However, in some cases, a strong dependence of ITS effects on promoter context was observed suggesting that promoters may have ‘allosteric’ abilities to modulate ITS effects. Large effects of ITS on promoter output and the observed interplay between promoter sequence and ITS effects suggests that the definition of bacterial promoter should include ITS sequence.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.

Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3′ untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3′ UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling.