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

• Published in: RNA (Cambridge) Vol. 4; no. 8; pp. 973 - 983
• Main Authors: GRENTZMANN, GUIDO, KELLY, PAUL J., LAALAMI, SOUMAYA, SHUDA, MASAHIRO, FIRPO, MATTHEW A., CENATIEMPO, YVES, KAJI, AKIRA
• Format: Journal Article
• Language: English
• Published: United States Cambridge University Press 01-08-1998
• Subjects: GTP Phosphohydrolase-Linked Elongation Factors - metabolism; Guanosine Triphosphate - metabolism; Hydrolysis; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Peptide Chain Termination, Translational; Peptide Elongation Factor G; Peptide Elongation Factors - metabolism; Peptide Termination Factors - metabolism; Ribosomes - metabolism; RNA, Transfer, Amino Acyl - metabolism; translation; RF1; GTPase; RRF; RF2
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1017/S1355838298971576

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.