Slow molecular recognition by RNA

Gleitsman, Kristin Rule; Sengupta, Raghuvir N.; Herschlag, Daniel

doi:10.1261/rna.062026.117

Cited by 43 publications

(64 citation statements)

References 85 publications

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“…However, the slow phase could instead represent an experimental artifact arising from a source such as non-uniform mixing across the ZMW chip upon delivery of RACK1-ybbR (Chen et al 2014). Regardless, the fast association rate we observed for RACK1 incorporation is similar to association rates observed for protein-protein (median ≈ 6.6 µM -1 s -1 ) and protein-RNA (median ≈ 6.3 µM -1 s -1 ) interactions (Schreiber 2002;Gleitsman et al 2017), and it provides a useful baseline rate for incorporation of a core ribosomal protein into mature human ribosomes.…”

Section: Rack1 Association With the 40s Ribosomal Subunitsupporting

confidence: 63%

RACK1 on and off the ribosome

Johnson

Lapointe

Wang

et al. 2019

Preprint

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Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

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Section: Rack1 Association With the 40s Ribosomal Subunitsupporting

confidence: 63%

RACK1 on and off the ribosome

Johnson

Lapointe

Wang

et al. 2019

Preprint

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“…In cells, steady-state patterns of RNA-protein interactions have been measured [3][4][5] . RBP binding and dissociation kinetics have only been determined in vitro 2,6 . For a small number of RBPs, equilibrium binding parameters measured in vitro correlate with steady-state binding patterns in cells 7,8 .…”

mentioning

confidence: 99%

The kinetic landscape of an RNA binding protein in cells

Sharma

Zagore

Brister

et al. 2020

Preprint

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4) Corresponding authors 2 ABSTRACT Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1 . The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNAprotein interactions 2 . However, these kinetics were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how previously inaccessible, kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.

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“…[12] Es gibt einige Kaskadenreaktionen im Primärstoffwechsel, bei der Interkonversion eines niedermolekularen Metaboliten in eines oder mehrere Produkte,die von allen Zellen vollzogen werden. In einem RNA-Welt-Szenario gingen ihnen katalytische RNAs voran, von denen heute noch Überbleibsel vorhanden sind, z.…”

Section: Enzymkaskadenunclassified

“…B. in den Ribonucleoprotein-Ribosomen, die die Proteinbiosynthese durchführen. [12] Es gibt einige Kaskadenreaktionen im Primärstoffwechsel, bei der Interkonversion eines niedermolekularen Metaboliten in eines oder mehrere Produkte,die von allen Zellen vollzogen werden. Beispielsweise werden durch die Tandemwirkung von Thiolase und Hydroxymethylglutaryl-CoA-Synthase drei Acetyl-CoA-Moleküle in den C 6 -Halbthioester HMG-CoA umgewandelt.…”

Section: Enzymkaskadenunclassified

“…[105] Dies ist eine faszinierende Kaskade mit Radikal-SAM-Enzymen. Es wird angenommen, dass etwa ein Drittel der mehr als 100 000 offenen Leserahmen, die als Radikal-SAM-Enzymei nP roteindatenbanken prognostiziert wurden, auch Vitamin B 12 Bisher sind noch keine Biosynthese-Analoga von ¾quivalenten der metallkoordinierten Alkenmetathese in der Enzymkatalyse bekannt. Andererseits produzieren Cyanobakterien Cylindrocyclophan F [107] nach einer vçllig anderen Kaskadenlogik.…”

Section: Angewandte Chemieunclassified

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Enzymkaskadenreaktionen in der Biosynthese

Walsh

Moore

2019

Angewandte Chemie

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Enzym‐vermittelte Kaskadenreaktionen sind weit verbreitet. Um einen Vergleich mit den mechanistischen Kategorisierungen der komplementären Kaskadenreaktionen in der organischen Synthese zu ermöglichen sowie um die gemeinsamen Grundlagen aufzuzeigen, erörtern wir hier vier Arten von Enzymkaskadenreaktionen: vermittelt durch nucleophile, elektrophile, pericyclische und radikalische Reaktionen. Zwei Subtypen von Enzymen, die radikalische Kaskaden erzeugen, befinden sich an den entgegengesetzten Enden der Abundanzskala von Sauerstoff: Eisen‐basierte Enzyme nutzen O2, um hochvalente Eisen‐Oxo‐Spezies zu erzeugen, die nichtaktivierte C‐H‐Bindungen in Substraten homolytisch spalten und Gerüstumlagerungen einleiten. Am anaeroben Ende spalten Enzyme reversibel S‐Adenosylmethionin (SAM) unter Bildung des 5′‐Desoxyadenosyl‐Radikals als starkes Oxidans, um die homolytische Spaltung von C‐H‐Bindungen in gebundenen Substraten auszulösen. Die letztgenannten Enzyme werden als Radikal‐SAM‐Enzyme bezeichnet. Die erstgenannten Enzyme kategorisieren wir als “verhinderte Oxygenasen”.

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Slow molecular recognition by RNA

Cited by 43 publications

References 85 publications

RACK1 on and off the ribosome

RACK1 on and off the ribosome

The kinetic landscape of an RNA binding protein in cells

Enzymkaskadenreaktionen in der Biosynthese

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