Mapping Functionally Important Motifs SPF and GGQ of the Decoding Release Factor RF2 to the Escherichia coli Ribosome by Hydroxyl Radical Footprinting

Abstract

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved 250 GGQ 252 motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, 205 SPF 207 , were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 Å only, a distance that is incompatible with the observed cleavage sites that are up to 100 Å apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.Termination of protein synthesis is signaled by the translocation of a stop rather than a sense codon into the A-site of the ribosome (1, 2). The stop codon is recognized by a protein, the polypeptide chain release factor (RF), 1 which triggers the hydrolytic release of the nascent polypeptide chain from the Psite-bound peptidyl-tRNA. Recognition of the stop codon by a protein rather than a tRNA molecule led Moffat and Tate (3) to propose the "tRNA analogue model," which describes the RF as a decoding molecule like tRNA. Class I RFs are the decoding factors (4). In bacteria such as Escherichia coli, there are two decoding RFs, RF1 (decodes UAG and UAA) and RF2 (decodes UGA and UAA). In eukaryotes, a single factor, eRF1, recognizes all three stop codons (5). With the exception of a universally conserved GGQ motif, there is no overall sequence homology between the eubacterial class I RFs and their eukaryotic counterparts (6). The properties of the class I RFs are remarkably similar to those of aminoacyl-tRNAs (reviewed in Ref. 7). RFs compete with suppressor tRNAs for binding to the A-site of the ribosome in response to specific recognition of the A-site codon (8, 9). This so-called anti-suppressor phenotype implies that tRNAs and RFs are cognate species in direct competition for stop codon binding. Both interact with the peptidyl-tRNA located at the ribosomal P-site, and both interact on the ribosome with translational G proteins (EF-Tu for aminoacyl-tRNA and RF3/eRF3 for the decoding RFs).In the tRNA analogue model (3), the decoding RF was proposed to consist of two functionally separate d...

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

“…The ribosomal region has been inferred previously to be relatively close to RF1 by hydroxyl radical footprinting [53] as well as to EF-G [54]. The cryoelectron microscopy model [30,31], hydroxyl radical footprinting [19,51] and genetic analysis [57] all confirmed the importance of this region of 23S rRNA as a site of potential interaction between factor and ribosome during translation termination.…”

“…The RF2 structure presented a puzzling paradox as to how the RF functioned on the ribosome. Further experiments have resolved this paradox by revealing that the molecule undergoes a highly significant conformational change [19,[30][31][32][33].…”

“…Methods [18,19] used for obtaining the novel data discussed here and brief strategies for already published experiments are described in Supplementary Information.…”

“…Specific cleavages were found only in the small subunit rRNA, with none identified in the rRNA of the large subunit. When mapped onto the three-dimensional model of the ribosome as determined by X-ray crystallography, they formed a ring delineating the DC of the ribosome [19] ( Figure 4B). Collectively, the data from the crosslinking studies and from the subdomain swapping experiments implied that the discriminatory motif was at the DC and must be at least near the mRNA, and the hydroxyl radical mapping provided compelling support for a major role of RF in termination codon recognition.…”

“…In addition, a single isolate of a fragment of helix 24 (green) was selected with RF1. Helix 24 contains the conserved 790 loop and its vicinity to RF1 and RF2 was earlier indicated by hydroxyl radical footprinting from specific sites on the factors [19,53,54].…”