| Autor: |
Anthony A. Paiva, Lee Huang, Liane M. Mende-Mueller, Ravi Kambampati, Carla B. Pellegrino, Kalpana Chakraburtty |
| Rok vydání: |
2000 |
| Předmět: |
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| Zdroj: |
Journal of Biological Chemistry. 275:16963-16968 |
| ISSN: |
0021-9258 |
| DOI: |
10.1074/jbc.m001157200 |
| Popis: |
Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 °C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH2-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH2-terminally, confirming their origin. The 50-kDa fragment (Ser2-Lys443) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser2-Lys668) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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