English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Dual-color pulse-chase ubiquitination assays to simultaneously monitor substrate priming and extension

Scott, D. C., & Schulman, B. A. (2019). Dual-color pulse-chase ubiquitination assays to simultaneously monitor substrate priming and extension. In Methods in Enzymology, 618: UBIQUITIN AND UBIQUITIN-LIKE PROTEIN MODIFIERS (pp. 29-48). Elsevier Academic Press.

Item is

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Scott, Daniel C.1, Author
Schulman, Brenda A.2, Author           
Affiliations:
1external, ou_persistent22              
2Schulman, Brenda / Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Max Planck Society, ou_2466699              

Content

show
hide
Free keywords: STRUCTURAL INSIGHTS; COMPLEX; E3; MECHANISM; LIGATION; LIGASES; CHAINS
 Abstract: Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the "pulse" reaction generates a thioester-linked enzyme similar to UB intermediate, while the "chase" monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2 similar to UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.

Details

show
hide
Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
 Pages: 20
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Methods in Enzymology, 618: UBIQUITIN AND UBIQUITIN-LIKE PROTEIN MODIFIERS
  Alternative Title : UBIQUITIN AND UBIQUITIN-LIKE PROTEIN MODIFIERS
Source Genre: Series
 Creator(s):
Affiliations:
Publ. Info: Elsevier Academic Press
Pages: - Volume / Issue: 618 (Kapitel 2) Sequence Number: - Start / End Page: 29 - 48 Identifier: -