Interaction of IF2 with the ribosomal GTPase-associated center during 70S initiation complex formation.

Initiation factor 2 (IF2) is a G-protein that plays a crucial role in the initiation of procaryotic protein synthesis, interacting directly with fMet-tRNAfMet, favoring its decoding in the P-site, and physically linking the 30S and 50S subunits in the 70S initiation complex (70SIC) (1–5). It shares a common binding locus on the ribosome, the GTPase associated center (denoted GAC), with other G-proteins utilized in protein synthesis, such as elongation factor G (EF-G). The GAC includes 23 S rRNA helices 42–44, the associated proteins L11, L10, and at least one L7/L12 protein (6, 7). Cryoelectron microscopy (cryo-EM) studies have indicated that both EF-G·GTP and IF2·GTP binding to the ribosome are accompanied by large conformational changes in the ribosome, and that, in both cases, further conformational changes in the ribosome·G-protein complex are seen following GTP hydrolysis (8 – 12). The N-terminal domain of L11 (L11-NTD) is a particularly mobile portion of the ribosome that, following GTP hydrolysis, approaches the G’ domain of EF-G (9, 13, 14). In earlier work utilizing single-turnover fluorescence resonance energy transfer (FRET) measurements, we determined that rapid movement of the G’ domain toward L11-NTD within the E. coli ribosome requires prior GTP hydrolysis and, via branching pathways, either precedes Pi release (major pathway) or occurs simultaneously with it (minor pathway) (15). In this latter work fluorescent groups were placed on the Cys 38 within L11-NTD and on a suitable residue within the G’ domain. Here we utilize a similar approach to determine whether there is a comparable movement toward L11-NTD of the G1-domain of IF2 (IF2 lacks a G’ domain) during 70SIC formation from 30S initiation complex (30SIC) and 50S subunit. The cryo-EM structures of 70S complexes containing fMet-tRNAfMet, mRNA and either IF2.GDPCP (a nonhydrolyzable analogue of GTP) or IF2·GDP (11) result in an estimated distance of 50 – 55 A between the α-carbons of residue 378 in IF2 and residue 38 in L11 (Figure 1), quite suitable for probing by measurement of FRET efficiency. Accordingly, we employ 50S subunits containing protein L11 labeled with Cy5 at position 38 (denoted L11Cy5) and B. stearothermophilus IF2 (Bst-IF2) labeled with Cy3 at position 378 (denoted Bst-IF2Cy3) (16). Figure 1 Distance between L11 residue 38 and IF2 residue 378 in a 70S.IF2 Substituting Bst-IF2 for Eco-IF2 has been shown to facilitate the characterization of translation intermediates, due to the higher stability of the complexes that Bst-IF2 forms with E. coli ribosomes and ribosomal subunits as compared with Eco-IF2 (16, 17). Such substitution is reasonable in view of substantial evidence that Bst-IF2 is functionally interchangeable with Eco-IF2 in E. coli protein synthesis. Thus, Bst-IF2 complements an E. coli infB null mutation in vivo (E. Caserta and C. Gualerzi, private communication). In addition, in vitro studies demonstrate the near equivalence of Eco-IF2, Bst-IF2, and Bst-IF2Cy3 in binary complex formation with E. coli fMet-tRNAfMet (18), in stimulating of AUG-dependent fMet-tRNAfMet binding to E. coli 30S subunits and 70S ribosomes (19) and in kinetic measures of 70SIC formation (17, 20 and work reported herein). We find that FRET efficiency increases as the 70S ribosomes formed initially from 30SIC and 50S subunits are transformed into the 70SIC, and that the rate of such increase depends on GTP hydrolysis, paralleling results obtained with EF-G. We further demonstrate that the increase in FRET efficiency can be used to monitor the fidelity function of the initiation factor IF3 during 70SIC formation.