Requirement for Ribosome‐Releasing Factor for the Release of Ribosomes at the Termination Codon

Abstract

With the use of 3H-labeled R17 amB2 phage RNA having an UAG codon at the seventh triplet of the coat cistron, release of the RNA from ribosomes at the termination codon was studied. The ribosome-releasing factor previously described was shown to stimulate the process of mRNA release at the termination codon. The stimulatory effect of the factor on this process was dependent on the termination factor (RF-1). GTP was required for this process and guanosine 5'-(P7y-methylene)triphosphate could not replace GTP. No apparent change of size of R17 RNA was observed during the release of the R17 RNA from the ribosomes. The ribosome-releasing factor is distinct from the known termination codon-specific factor such as RF-1.In the preceding publications [l -31 it was demonstrated that elongation factor G(EF-G), a previously unknown factor (tentatively named ribosome-releasing factor), and GTP were necessary for release of ribosomes from naturally occurring polysome which had been treated with puromycin. The ribosomereleasing factor has been purified to electrophoretic homogeneity and has a molecular weight of approximately 18000. It was proposed that this factor may play some role in the release of ribosomes from messenger RNA at the termination point of each cistron.In this communication, evidence is presented that ribosome-releasing factor can function for the release of ribosomes at the termination codon as well as other points in the messenger RNA. With the use of amB, R17RNA which has an amber codon at the seventh triplet of coat cistron [4-61, it was demonstrated that the factor catalyzed the release of mRNA from ribosomes at the amber codon. The released RNA was not fragmented during the release from ribosomes. Definirion. A,, unit, the quantitity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm pathlength cell. EXPERIMENTAL PROCEDURE Preparation of Various FactorsThe ribosome-releasing factor was purified to electrophoretic homogeneity as described previously [l]. The crude soluble fraction of Escherichia coli containing the termination factor RF-1, was prepared free of ribosome-releasing factor as follows. The ribosome-free supernatant fluid (1 1, 9.26 g) prepared as described previously (step one of the purification of the factor by Hirashima and Kaji [I]). The protein fraction precipitated between 45 and 75 % saturation of ammonium sulfate, collected by centrifugation at 8500 rev./min for 15 min in a Sorvall GSA rotor, dissolved in 80ml of 10mM Tris-HC1 (pH 7.8), l.OmM magnesium acetate, 20 mM ammonium chloride, 0.5 mM dithiothreitol, 5 % glycerol, and dialyzed against the same buffer overnight. Insoluble material was removed by centrifugation and the solution (80 ml) containing 5.33 g of protein was placed on a DEAEcellulose column (2.6 x 86 cm), which had been equilibrated with the same buffer. The loaded column was washed with the same buffer. The ribosome-releasing factor was eluted during this washing process. The proteins retained on the column were...

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

“…The GTPase activity of RF3 (Table 1) was very much reminiscent of the GTPase activity of EF-G (Conway & Lipmann, 1964)+ EF-G has been shown to be required for ribosome recycling in vitro, previously defined as the dissociation of mRNA and tRNA from the terminated ribosome, in the presence of RRF and GTP (Hirashima & Kaji, 1972)+ We therefore asked whether RF3 might be able to release mRNA from the ribosome in the presence of RRF and GTP (Table 2)+ We used an assay that shows release of the 32 P-labeled UUC AUG UAA minimessenger RNA (*mRNA) (Table 2A), 35 Sfmet (*fmet), as well as nonhydrolyzed 35 S-fmettRNA f met (*fmet-tRNA) (Table 2B), in the presence of RF1 or RF2, RF3 or EF-G, RRF, and GTP+ Using the minimessenger RNA, we were able to reproduce previously published results on release of naturally occurring mRNA dependent on EF-G and RRF in the presence of GTP (Ogawa & Kaji, 1975) (Table 2A, line 1, compare to line 6)+…”

“…To establish that RF3 replaces the role of EF-G in the release of ribosomes by RRF, RF3 dose-response curves for mRNA release were determined (Fig+ 4)+ Release of mRNA increased with the amount of added RF3 in complete absence of EF-G+ This establishes that RRF-dependent mRNA release can use RF3 instead of EF-G+ To verify termination dependence of ribosome recycling by RF3 and RRF, we established the RF3 dose-response curves at lower concentration of RRF than in initial experiments (Table 2), and in the absence or presence of release factor RF2+ Under these conditions, release of mRNA from ribosomes was dependent on the presence of RF2+ Even at concentrations of RF3 (0+22 units) allowing maximal ribosome recycling, termination-independent recycling was not significant+ To further confirm release of mRNA by RRF from ribosomes in the presence of RF3, a doseresponse curve of mRNA release, regarding RRF, was established (Fig+ 5)+ Again, mRNA release increased with added amounts of RRF+ (Ogawa & Kaji, 1975)+ Released f-met and mRNA are expressed in 1/100 pmol+ Backgrounds of free fmet (50 fmol) and free mRNA (150 fmol) in absence of factors were subtracted+ Standard deviations were between 5 and 15%+ …”

“…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)+…”

“…f[ 35 S]met-tRNA f met {messenger{ribosome complex was prepared as described (Grentzmann & Kelly, 1997), incubating 50 pmol of 70S ribosomes with 50 pmol charged tRNA in the presence of 250 pmol RNA oligonucleotide for 20 min in 20 mM Tris, pH 7+2, 150 mM NH 4 Cl, and 30 mM Mg(OAc) 2 at 30 8C in a final volume of 50 mL+ Termination kinetics were run in 80 mM Tris, pH 7+2, 100 mM KCl, and 8 mM Mg(OAc) 2 as described at 24 8C or 30 8C (Caskey et al+, 1971)+ Ribosome complex was added at 0+5 pmol per 10 mL reaction volume+ GTP, when added, was 160 mM+ RRF in pmol, RF1 and RF2 in picounits (Caskey et al+, 1971), RF3 in units (Grentzmann et al+, 1994) (Ogawa & Kaji, 1975) at 30 8C+ RF-1, -2 and -3, EF-G, RRF, and 160 mM GTP were added as indicated+ When establishing conditions for this assay, we found that addition of 0+2 mM phosphoenolpyruvate and 3 mg of pyruvate kinase, which significantly enhances ribosome recycling in the presence of EF-G, was not essential when using RF3 (data not shown)+ To minimize the risk of mRNA release due to contamination of RF3 fractions by EF-G, we performed our RF3 experiments without the above-mentioned GTP-generating system+ Hydrolyzed formyl-methionine was counted after ethyl acetate extraction+ Released mRNA was counted after filtration through Microcon 10 filters (Amicon) at 4 8C+ Released nonhydrolyzed fmettRNA fmet was estimated by counting the 35 S difference after repurification of reaction mixtures on S-300 spun columns and subtracting values for hydrolyzed formyl-methionine+ Assays were repeated two to four times with independent complex preparations+ ACKNOWLEDGMENTS RF1-and RF2-overexpression plasmids were a kind gift from Dr+ W+P+ Tate+ We thank Dr+ Y+ Kaziro for providing EF-G and …”

“…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+…”