Biology subjects

Feigon, J.

Publications and source records attributed to Feigon, J..

5 recordsLinked to original sources

bioRxiv2026

Cryo-EM Structure of a 95-Basepair Double-Stranded DNA Minicircle at 5.3 A Resolution

Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-basepair minicircle (dsMC95) at a 5.3 [A] resolution. dsMC95 forms a closed ring as designed and no local deformation is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits an in-plane ellipticity of 1.13 and an out-of-plane displacement of 15{degrees}, with differences in out-of-plane displacements observed between the two half-segments. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 [A] to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and may inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.

biophysics↗

bioRxiv2024

HEXIM1 homodimer binds two sites on 7SK RNA to release autoinhibition for P-TEFb inactivation

Hexim proteins are key RNA-dependent regulators of eukaryotic transcription through 7SK-dependent sequestration and inactivation of the kinase P-TEFb (Cdk9-CyclinT1/2) in the 7SK RNP. P-TEFb activity drives release of RNA polymerase II from promoter-proximal pausing for eukaryotic and HIV-1 transcription. The molecular mechanism by which 7SK binding overcomes an intrinsic Hexim autoinhibition for subsequent P-TEFb inactivation has remained unresolved. Here, using NMR and biophysical methods we demonstrate that Hexim1 homodimer engages two high-affinity sites on 7SK RNA. This dual-site binding triggers a conformational rearrangement in Hexim1s disordered central region that unmasks the Cdk9-binding site, which is otherwise sequestered within an intermonomer dimer interface. These findings reveal how Hexim autoinhibition dictates its specificity for 7SK RNA and prevents premature P-TEFb inhibition in the absence of 7SK, thereby providing a mechanistic understanding of Hexim/P-TEFb assembly into the 7SK RNP and further considerations for understanding Hexim-Tat competition during viral transcription.

biophysics↗

bioRxiv2024

How short peptides can disassemble ultra-stable tau fibrils extracted from Alzheimers disease brain by a strain-relief mechanism

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimers disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC disassembles tau fibrils from AD brains (AD-tau) into benign segments with no energy source present beyond ambient thermal agitation. This disassembly by a short peptide was unexpected, given that AD-tau is sufficiently stable to withstand disassembly in boiling SDS detergent. To consider D peptide-mediated disassembly as a potential therapeutic for AD, it is essential to understand the mechanism and energy source of the disassembly action. We find assembly of D-peptides into amyloid-like fibrils is essential for tau fibril disassembly. Cryo-EM and atomic force microscopy reveal that these D-peptide fibrils have a right-handed twist and embrace tau fibrils which have a left-handed twist. In binding to the AD-tau fibril, the oppositely twisted D-peptide fibril produces a strain, which is relieved by disassembly of both fibrils. This strain-relief mechanism appears to operate in other examples of amyloid fibril disassembly and provides a new direction for the development of first-in-class therapeutics for amyloid diseases.

neuroscience↗

bioRxiv2021

Structure of telomerase-bound CST with Polymerase α-Primase

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3-overhang of the G-strand and a group of proteins called shelterin1,2. Among these, TPP1 and POT1 associate with the 3-overhang, with POT1 binding the G-strand3 and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT)4. The ends of the telomeric DNA are replicated and maintained by telomerase5, for the G-strand, and subsequently DNA Polymerase -Primase6,7 (PolPrim), for the C-strand8. PolPrim is stimulated by CTC1-STN1-TEN1 (CST)9-12, but the structural basis of both PolPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolPrim, as well as of PolPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex--telomerase core RNP, p50/TPP1, CST and PolPrim--that provides unprecedented insights into CST and PolPrim recruitment and handoff between G-strand and C-strand synthesis.

molecular biology↗

bioRxiv2019

Structure of S. pombe telomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins

La related proteins group 7 (LARP7) are a class of RNA chaperones that bind the 3ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In ciliates, a LARP7 protein (p65 in Tetrahymena) is a core component of telomerase, an essential ribonucleoprotein complex that maintains the DNA length at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase (TERT). Although a LARP7 as a telomerase holoenzyme component was initially thought to be specific to ciliate telomerases, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. There is also evidence that human Larp7 associates with telomerase. LARP7 proteins have conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and hLarp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix 3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.\n\nHighlightsO_LIThe structure of the S. pombe LARP7 Pof8 C-terminal domain is an xRRM.\nC_LIO_LICiliates, human, and fission yeast contain LARP7 proteins with xRRMs involved in telomerase biogenesis.\nC_LIO_LIWith three examples of xRRM structures, we refine the definition of xRRM.\nC_LI

biophysics↗