Timing of GTP binding and hydrolysis by translation termination factor RF3

Abstract

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.

RF3:GTP promotes rapid dissociation of the class 1 termination factor

“…1A). Consistent with the previously published binding affinity of RF3 for GDP (Zavialov et al 2001;Peske et al 2013) and the observation that RF3 copurifies with GDP, the K D for GDP binding to RF3 is relatively tight (13 nM ± 9 nM) (Fig. 1A).…”

“…Using a reconstituted E. coli translation system, we find that the K D s for GDP and GTP are more closely matched (within fourfold) than previously observed (these data agree with a report published while this manuscript was under review [Peske et al 2013]). Also, as previously reported (Zavialov et al 2001;Peske et al 2013), the off-rates for bound nucleotide (GTP or GDP) are slow, consistent with the need for a GEF to allow translation to proceed at physiological rates. We further show that RF3:GDPNP has considerably higher affinity than RF3:GDP for ribosome termination complexes and that the nonrotated state is slightly preferred.…”

“…Other studies have similarly attempted to remove bound nucleotide (Pallesen et al 2013;Peske et al 2013). Most recently, Wintermeyer and colleagues demonstrated by FPLC the loss of bound nucleotide but found that RF3 activity was then diminished (Peske et al 2013). In the studies below, we characterize the binding of functional properties of both naturally isolated RF3:GDP as well as the EDTAtreated RF3:GDP sample and find their properties to be indistinguishable.…”

“…1). Other studies have similarly attempted to remove bound nucleotide (Pallesen et al 2013;Peske et al 2013). Most recently, Wintermeyer and colleagues demonstrated by FPLC the loss of bound nucleotide but found that RF3 activity was then diminished (Peske et al 2013).…”

Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

“…Unlike sense codons, which are decoded by tRNAs, stop codons are decoded by proteinaceous release factors (RFs). 2 There are two classes of RFs: class 1 factors that recognize the stop codon and promote release of the nascent peptide, and class 2 factors that appear to fulfill distinct functions in bacteria and eukaryotes (1)(2)(3)(4). Bacteria possess two class 1 RFs with overlapping specificity: RF1 recognizes UAG and UAA, whereas RF2 recognizes UGA and UAA.…”