Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2.
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 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A 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.
Scarlett
Debbie-Jane G
DJ
Department of Biochemistry and Centre for Gene Research, University of Otago, P. O. Box 56, Dunedin, New Zealand.
McCaughan
Kim K
KK
Wilson
Daniel N
DN
Tate
Warren P
WP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009154:Mutation; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism; D010458:Peptidyl Transferases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D023302:Protein Footprinting / Q000379:methods*; D012270:Ribosomes / Q000737:chemistry*
D010454:Peptide Termination Factors; C041460:peptide chain termination release factor 2; D017665:Hydroxyl Radical; D010458:Peptidyl Transferases