Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex.

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3-Cy5 distance to ∼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper ß'-clamp while downstream DNA contacts the top of the ß-pincer in I1E. Converting I1E to I1M (∼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on ß') away from downstream DNA (on ß) to increase the Cy3-Cy5 distance by ∼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.

Thyroid hormone signaling specifies cone photoreceptor subtypes during eye development: Insights from model organisms and human stem cell-derived retinal organoids.

Cones are the color-detecting photoreceptors of the vertebrate eye. Cones are specialized into subtypes whose functions are determined by the expression of color-sensitive opsin proteins. Organisms differ greatly in the number and patterning of cone subtypes. Despite these differences, thyroid hormone is an important regulator of opsin expression in most vertebrates. In this chapter, we outline how the timing of thyroid hormone signaling controls cone subtype fates during retinal development. We first examine our current understanding of cone subtype specification in model organisms and then describe advances in human stem cell-derived organoid technology that identified mechanisms controlling development of the human retina.

R-loop induced G-quadruplex in non-template promotes transcription by successive R-loop formation.

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.

G-Quadruplex and Protein Binding by Single-Molecule FRET Microscopy.

G-quadruplex (G4) is a non-canonical nucleic acid structure that arises from the stacking of planar G-tetrads, stabilized by monovalent cations. G4 forming sequences exist throughout the genome and G4 structures are shown to be involved in many processes including DNA replication and gene expression. The single-molecule total internal reflection fluorescence (TIRF) microscopy has been employed to study G4 structure formation and protein binding interactions. Here, we describe methods by which we tested the folding and unfolding of G-quadruplexes structure and studied the dynamics of its interaction with POT1 protein. The methods presented here can be applied to study other putative G4 sequences and potential binding partners.