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Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

Published online by Cambridge University Press:  01 August 1998

GUIDO GRENTZMANN ,
PAUL J. KELLY ,
SOUMAYA LAALAMI ,
MASAHIRO SHUDA ,
MATTHEW A. FIRPO ,
YVES CENATIEMPO  and
AKIRA KAJI
GUIDO GRENTZMANN
Affiliation:
Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112, USA
PAUL J. KELLY
Affiliation:
University of Utah, Salt Lake City, Utah 84112, USA
SOUMAYA LAALAMI
Affiliation:
ESA-CNRS 6031, Institut de Biologie Moléculaire et d'Ingénierie Génétique, Université de Poitiers, 86000 Poitiers, France
MASAHIRO SHUDA
Affiliation:
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
MATTHEW A. FIRPO
Affiliation:
Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112, USA
YVES CENATIEMPO
Affiliation:
ESA-CNRS 6031, Institut de Biologie Moléculaire et d'Ingénierie Génétique, Université de Poitiers, 86000 Poitiers, France
AKIRA KAJI
Affiliation:
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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Abstract

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets.

We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

Keywords

GTPaseRF1RF2RRFtranslation
Type
Research Article
Information
RNA , Volume 4 , Issue 8 , August 1998 , pp. 973 - 983
Copyright
© 1998 RNA Society

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