Biology subjects

Wasserman, M. R.

Publications and source records attributed to Wasserman, M. R..

4 recordsLinked to original sources

bioRxiv2022

Didemnin B and ternatin-4 inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. Two structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common allosteric site on eEF1A, didemnin B and ternatin-4 trap eEF1A in its GTPase-activated conformation, preventing aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings highlight the potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.

biochemistry↗

bioRxiv2021

ORC binds and remodels nucleosomes to specify MCM loading onto DNA

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c.. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with new analysis of genome-wide ORC binding, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

biophysics↗

bioRxiv2018

Dynamics of Cas10 Govern Discrimination between Self and Nonself in Type III CRISPR-Cas Immunity

Adaptive immune systems are required to accurately distinguish between self and nonself in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNAs that recognize complementary RNA molecules to trigger the degradation of both the target transcript and its template DNA. These systems can broadly eliminate foreign targets with multiple mutations, but still effectively curb immunity against the host. The molecular basis for these unique features remains unknown. Here we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10--the DNase effector of the complex--displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Single-stranded DNA promotes Cas10s occupancy at a selected set of conformational states, which is also sensitively modulated by target mutations and predictive of CRISPR interference activity. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self/nonself discrimination and target specificity.

biophysics↗

bioRxiv2018

Replisome preservation by a single-stranded DNA gate in the CMG helicase

The eukaryotic replicative helicase CMG is assembled at replication origins and is thought to remain topologically closed until termination. Upon encountering a lesion, CMG must vacate a stalled fork to allow DNA repair. However, the fate of CMG under these stress conditions remains unclear. Here, using correlative single-molecule fluorescence and force microscopy, we show that when uncoupled from a DNA polymerase, CMG opens a single-stranded (ss) DNA gate to traverse a forked junction and reside on double-stranded (ds) DNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA for continued fork progression. The accessory protein Mcm10 is required for robust ssDNA gating. These results reveal an Mcm10-induced pathway that preserves CMG on DNA and allows it to access a repaired fork for swift replication recovery.

biophysics↗