Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2

Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide’s 3’-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Single-molecule analysis of transcription initiation in archaea and eukaryotes

Transcription is the transfer of information stored in the DNA genome into RNA and an essential process for cellular life. In all three domains of life, transcription is carried out by structurally conserved RNA polymerases (RNAP). archaea employ only a single RNAP while eukaryotes have evolved three functionally distinct variants, RNAP I, II and III. Transcription is a cyclic process divided into initiation, elongation and termination phases. During transcription initiation, RNAPs are recruited to the promoter region of their target genes by transcription factors (TF) that recognize sequence elements in the promoter DNA, leading to formation of a pre-initiation complex (PIC). In archaeal and all eukaryotic transcription systems, initiation relies on the TATA-binding protein (TBP) and a TFIIB-like factor. TFIIIB, the central initiation factor of the human RNAP III system is composed of TBP, the TFIIB-related factors Brf1 or Brf2 and the RNAP III-specific factor Bdp1. At TATA-box-containing promoters, TFIIIB alone can recruit RNAP III. In contrast, RNAP II recruitment can be achieved by TBP and TFIIB. How TFIIIB dynamically assembles at the promoter and the role of Bdp1 as an additional required factor are still not well understood. Furthermore, TFIIIB forms highly stable complexes on DNA that can persist for multiple rounds of transcription. How transcription factors are affected by mechanical forces exerted on DNA by cellular processes like transcription and DNA compaction is mostly unexplored. The archaeal transcription machinery resembles the eukaryotic RNAP II system, but gene regulation is performed by Bacteria-like transcriptional regulators. In Pyrococcus furiosus, the TFB recruitment factor 1 (TFB-RF1) can recruit TFB to promoters with a weak B recognition element (BRE) by binding to a conserved upstream promoter element. The molecular mechanism and potential conformational changes in the DNA are still elusive. Many archaea evolved paralogs of TBP and TFB that are involved in stress response. In Sulfolobus acidocaldarius, TFB2, a paralog of TFB is expressed in a cell cycle dependent manner. However, its function in the context of transcription is still unexplored. Assembly of TFs at the promoters is highly dynamic and involves TBP-induced bending of the promoter DNA by approximately 90°. This conformational change can be detected by Förster resonance energy transfer (FRET), a distance-dependent non-radiative process that can report on distance changes in the 1–10 nm regime with high time resolution. In this work, FRET measurements are performed at the single-molecule level, which allows direct observation of different conformational states of individual initiation complexes in real-time. Furthermore, force measurements were performed with a DNA origami-based nanodevice that can exert constant force in the piconewton range on a double stranded DNA fragment. To facilitate FRET experiments, a total internal reflection fluorescence (TIRF) microscope was constructed. In this work, TIRF and confocal fluorescence microscopy were used to study the molecular mechanisms underlying human and archaeal transcription initiation. The stepwise assembly of TFIIIB at the U6 snRNA promoter was monitored, revealing transient DNA binding of TBP on the millisecond timescale. Addition of Brf2 and Bdp1 stabilized dynamic DNA/TBP complexes. Force measurements revealed that binding of human TFs to DNA under mechanical strain is impaired. The complete TFIIIB complex can bind to DNA at forces up to 6.3 pN, whereas binding of TBP and TBP/Brf2 was strongly reduced at 2.6 pN and 6.3 pN, respectively. A comparison with the RNAP II-specific TFIIB and TFIIA provides evidence that the cage-like structure of TFIIIB confers superior resistance to mechanical force. S. acidocaldarius TFB2 cannot stabilize a bent promoter DNA/TBP complex and is thus not functional as a transcription initiation factor. Instead, six-fold molar excess of TFB2 could destabilize a DNA-bound TBP/TFB complex, suggesting a regulatory role for TFB2. TFB-RF1 can enhance TFB recruitment at promoters with weak or consensus BREs to similar degree without inducing conformational changes in the DNA. However, formation of a bent promoter DNA complex is slower at promoters with a consensus BRE. TFB-RF1 likely acts as a secondary binding site for TFB. DNA origami-based force spectroscopy further demonstrated force-dependent DNA bending of archaeal TFs. Methanocaldococcus jannaschii TBP can bend DNA alone whereas P. furiosus requires TBP and TFB. For both systems, low DNA bending efficiency was observed at 6.2 pN force. Taken together, this work highlights conserved mechanisms employed by archaeal and eukaryotic transcription systems to modulate the stability of the TBP/TFIIB-like factor complex that nucleates PIC formation, thereby enabling control over transcription initiation

Mechanistic insights into Lhr helicase function in DNA repair

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the `parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication

Structural and functional insights into the fly microRNA biogenesis factor Loquacious

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Logs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Logs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Logs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Logs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Funder: Ludwig Maximilians University Munich | Center for NanoScience, Ludwig-Maximilians-Universität Mnchen (CeNS, LMU); doi: https://doi.org/10.13039/501100007153Funder: Alexander von Humboldt-Stiftung (Alexander von Humboldt Foundation); doi: https://doi.org/10.13039/100005156Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology