Kinetics and thermodynamics of RRF, EF-G, and thiostrepton interaction on the Escherichia coli ribosome.
Ribosome recycling factor (RRF) and elongation factor-G (EF-G) are jointly essential for recycling bacterial ribosomes following termination of protein synthesis. Here we present equilibrium and rapid kinetic measurements permitting formulation of a minimal kinetic scheme that accounts quantitatively for RRF and EF-G interaction on the Escherichia coli ribosome. RRF and EF-G (a) each form a binary complex on binding to a bare ribosome which undergoes isomerization to a more stable complex, (b) form mixed ternary complexes on the ribosome in which the affinity for each factor is considerably lower than its affinity for binding to a bare ribosome, and (c) each bind to two sites per ribosome, with EF-G having considerably higher second-site affinity than RRF. Addition of EF-G to the ribosome-RRF complex induces rapid RRF dissociation, at a rate compatible with the rate of ribosome recycling in vivo, but added RRF does not increase the lability of ribosome-bound EF-G. Added thiostrepton slows the initial binding of EF-G, and prevents both formation of the more stable EF-G complex and EF-G-induced RRF dissociation. These findings are relevant for the mechanism of post-termination complex disassembly.
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Hyuk-Soo
HS
Seo
Michael
M
Kiel
Dongli
D
Pan
V Samuel
VS
Raj
Akira
A
Kaji
Barry S
BS
Cooperman
D003374:Coumarins; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012269:Ribosomal Proteins; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013883:Thiostrepton / Q000494:pharmacology*
D003374:Coumarins; D009711:Nucleotides; D020653:Peptide Elongation Factor G; D011506:Proteins; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D013883:Thiostrepton