Ribosome recycling by ribosome recycling factor (RRF) ? An important but overlooked step of protein biosynthesis

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

“…Model for RF3 function in translational termination+ I, complex formation RF1 binds stronger to the termination complex than RF2 (Goldstein et al+, 1970b;Grentzmann et al+, 1995), the thin double-headed arrow indicates lower binding affinity for RF2; II, terminal peptidyl-tRNA hydrolysis; III, ribosome recycling; IV, termination and ribosome recycling in absence of RF3 (Janosi et al+, 1996)+ RF3 is not essential (Grentzmann et al+, 1994;Mikuni et al+, 1994) and termination occurs in the absence of RF3 (Caskey et al+, 1971)+ EF-G has been shown to catalyze ribosome recycling in vitro, in the presence of RRF and GTP (Ogawa & Kaji, 1975;Janosi et al+, 1996) (Fig+ 7, IV)+ At present, we do not know whether EF-G, RF3, or both factors function for disassembly of the termination complex by RRF in vivo+ It is possible that EF-G can replace RF3 in this function in vivo in an RF3 Ϫ mutant or in organisms like Mycoplasma genitalium, which do not contain an RF3 gene (Fraser et al+, 1995)+ On the other hand, it is also possible that the function of RF3 is limited to the termination stop and the ribosome recycling step may as well be catalyzed by EF-G and RRF, as proposed previously (Janosi et al+, 1996)+…”

“…After translational termination by RF1 or RF2, the termination complex must be disassembled in order to recycle ribosomes, tRNA, mRNA, and release factors+ In vitro release of tRNA and mRNA from the ribosome is catalyzed by elongation factor EF-G and the ribosome recycling factor (RRF, formerly called ribosome releasing factor) in presence of GTP (Hirashima & Kaji, 1972;Ogawa & Kaji, 1975;Janosi et al+, 1996)+ RRF is essential for cell growth (Janosi et al+, 1994)+ We tested whether RF3 could replace EF-G GTPase activity to allow ribosome recycling by RRF+ In this paper, we report a GTPase activity with RF3, which is only dependent on ribosomes, much comparable to the GTPase activity of EF-G+ RF3 stimulates RF1-catalyzed termination dependent on GTP, whereas termination with RF2 is stimulated independently of the presence of GTP+ RF3 can replace EF-G in the in vitro mRNA release from the ribosome by RRF, dependent on GTP+ Excess of RF3 and RRF can result in decomposition of the translation complex, independent of previous termination by RF1 or RF2+…”

Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C‐terminal modulator element

“…One contradiction that remains to be resolved is whether RRF is able to dissociate the mRNA or not. Pavlov et al [7] have shown no release of the mRNA by RRF in vitro, and there are claims against the early work by Kaji and colleagues (review in [2]). …”

Functional mapping of ribosome-contact sites in the ribosome recycling factor: A structural view from a tRNA mimic

“…Translation termination proceeds in two sequential steps: The release of nascent polypeptides at stop codons and the disassembly of the posttermination complex + In bacteria, the ribosome recycling factor (RRF), in concert with the elongation factor EF-G, plays a main role in the second step for the next round of protein synthesis (for a review, see Janosi et al+, 1996)+ After release of nascent polypeptides by polypeptide release factors RF1 and RF2, the ribosomal P-site and A-site remain occupied with a deacylated tRNA and RF1 or RF2 protein+ Another class of bacterial release factor, RF3, accelerates the dissociation of RF1 and RF2 from the ribosome in a GTP-dependent manner, and RF3 is also released from the ribosome upon GTP hydrolysis (Freistroffer et al+, 1997;Pavlov et al+, 1997)+ These processes leave the posttermination complex with mRNA, deacylated tRNA in the P-site, and the empty A-site, which is believed to be a substrate for RRF in concert with EF-G (Hirashima & Kaji, 1972)+ The crystal structure of RRF has recently been solved to 2+55, 2+3, and 2+6 Å resolution by three groups using RRF proteins from Thermotoga maritima (Selmer et al+, 1999), Escherichia coli (Kim et al+, 2000), and Thermus thermophilus (Toyoda et al+, 2000)+ These three molecules are composed of two domains, domain 1 and domain 2, bridged by two loops (a hinge), and superimpose almost perfectly with tRNA Phe except for the amino acid-binding 39 end+ Selmer et al+ (1999) have proposed that RRF is a near perfect tRNA mimic to explain the mechanistic disassembly of the posttermination ribosomal complex+ They speculate that RRF binds to the A-site of the ribosome and that EF-G translocates RRF from the A-to the P-site and deacylated tRNA from the P-to the E-site of the ribosome in a GTP-dependent manner, where it would dissociate rapidly+ RRF, however, is architecturally different from tRNA in that the hinge of RRF forms a flexible "gooseneck" elbow, whereas the elbow of tRNA is rigid, and this flexibility of RRF is vital for its function (Toyoda et al+, 2000)+ Moreover, the model by Selmer et al+ (1999) is not consistent with the biochemical findings of Karimi et al+ (1999), which show, first, that RRF and EF-G split the ribosome into subunits in a reaction that requires GTP hydrolysis and, second, that the initiation factor IF3 is required for the removal of deacylated tRNA from the P-site of the 30S particle+ Thus the mechanistic significance of a tRNA mimic by RRF remains to be tested+ In fact, very little is known about the structureand-function relationship of RRF, which piqued our interest in a functional mapping of the ribosome-binding site in RRF+…”