Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2.
The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg(2+) and 37 degrees C. Filter binding assays conducted in the absence of magnesium ions on fMet-tRNA(f), eosin-Met-tRNA(f), and cascade yellow-Met-tRNA(f) confirmed the previously reported value for the dissociation constant of fMet-tRNA(f) of about 1 microM and placed the binding constants for the two fluorophore derivatives about three-fold higher. Binding of the fluorophore-Met-tRNA(f) species to salt-washed ribosomes showed a more significant decrease compared to fMet-tRNA(f). Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF-2 was observed in each case. All ribosome-bound cascade yellow-Met-tRNA(f) and eosin-Met-tRNA(f) were as puromycin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides.
McIntosh
B
B
Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
Ramachandiran
V
V
Kramer
G
G
Hardesty
B
B
D018387:Codon, Initiator; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes; D008301:Maleimides; D008851:Micropore Filters; D010452:Peptide Biosynthesis; D010442:Peptide Chain Initiation, Translational; D011691:Puromycin / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012492:Salts
D018387:Codon, Initiator; D015852:Eukaryotic Initiation Factor-2; D005456:Fluorescent Dyes; D008301:Maleimides; D012358:RNA, Transfer, Met; D012492:Salts; C024988:tRNA, formylmethionine-; D011691:Puromycin; C033531:eosin maleimide; C003944:N-(3-pyrene)maleimide; D004801:Eosine Yellowish-(YS)