IF2 Interaction with the Ribosomal GTPase-Associated Center During 70S Initiation Complex Formation

摘要

Addition of an E. 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). IF2 and 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 Pi release, paralleling movement of EF-G following its binding to the ribosome [Seo 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 non-canonical initiation codon during 70SIC formation. We employ B. 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 suggests that Bst-IF2·GDP is more difficult to displace from the GAC than E. coli IF2.GDP.