A quantitative kinetic scheme for 70 S translation initiation complex formation.
Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50 S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and fMet-tRNA(f)(Met). According to this scheme, an initially formed labile 70 S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30 S and 50 S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA(fMet), which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA(fMet). The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the non-hydrolyzable analog GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70 S formation, but perturbs fMet-tRNA(fMet) positioning in the P-site. In contrast, thiostrepton severely retards stable 70 S formation, but allows normal binding of fMet-tRNA(fMet)(prf20) to the P-site.
Grigoriadou
Christina
C
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Marzi
Stefano
S
Kirillov
Stanislas
S
Gualerzi
Claudio O
CO
Cooperman
Barry S
BS
D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007700:Kinetics; D010442:Peptide Chain Initiation, Translational; D039881:Prokaryotic Initiation Factor-2 / Q000378:metabolism; D039665:Prokaryotic Initiation Factor-3 / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D012270:Ribosomes / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C025207:eIF-2 Met-tRNA(f)(met) GDPCP; D020558:GTP Phosphohydrolases