Interaction of IF2 with the ribosomal GTPase-associated center during 70S initiation complex formation.
Addition of an Escherichia coli 50S subunit (50S(Cy5)) containing a Cy5-labeled L11 N-terminal domain (L11-NTD) within the GTPase-associated center (GAC) to an E. coli 30S initiation complex (30SIC(Cy3)) containing Cy3-labeled initiation factor 2 complexed with GTP leads to rapid development of a FRET signal during formation of the 70S initiation complex (70SIC). Initiation factor 2 (IF2) and elongation factor G (EF-G) induce similar changes in ribosome structure. Here we show that such similarities are maintained on a dynamic level as well. Thus, movement of IF2 toward L11-NTD after initial 70S ribosome formation follows GTP hydrolysis and precedes P(i) release, paralleling movement of EF-G following its binding to the ribosome [Seo, H., et al. (2006) Biochemistry 45, 2504-2514], and in both cases, the rate of such movement is slowed if GTP hydrolysis is prevented. The 30SIC(Cy3):50S(Cy5) FRET signal also provides a sensitive probe of the ability of initiation factor 3 to discriminate between a canonical and a noncanonical initiation codon during 70SIC formation. We employ Bacillus stearothermophilus IF2 as a substitute for E. coli IF2 to take advantage of the higher stability of the complexes it forms with E. coli ribosomes. While Bst-IF2 is fully functional in formation of E. coli 70SIC, relative reactivities toward dipeptide formation of 70SICs formed with the two IF2s suggest that the Bst-IF2.GDP complex is more difficult to displace from the GAC than the E. coli IF2.GDP complex.
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Haiou
H
Qin
Christina
C
Grigoriadou
Barry S
BS
Cooperman
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D001411:Geobacillus stearothermophilus; D006868:Hydrolysis; D008027:Light; D010442:Peptide Chain Initiation, Translational; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism*; D012542:Scattering, Radiation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D021122:Protein Subunits; D012269:Ribosomal Proteins; D020558:GTP Phosphohydrolases