Discrimination between mono- and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E.
Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m(7)GTP or m(3)(2,2,7)GTP. Caenorhabditis elegans also expresses five isoforms of the cap-binding protein eIF4E. Some isoforms (e.g. IFE-3) bind to m(7)GTP-Sepharose exclusively, whereas others (e.g. IFE-5) bind to both m(7)GTP- and m(3)(2,2,7)GTP-Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE-3 and IFE-5, based on the known structure of mouse eIF4E-1. We then substituted amino acid sequences of IFE-5 with homologous sequences from IFE-3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE-5 to essentially that of IFE-3. Molecular dynamics simulations suggested that the width and depth of the cap-binding cavity were larger in IFE-5 than in IFE-3 or the N64Y/V65L variant, supporting a model in which IFE-3 discriminates against m(3)(2,2,7)GTP by steric hindrance. Furthermore, the affinity of IFE-5 (but not IFE-3) for m(3)(2,2,7)GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys-122 and Cys-126. Thus, translation of m(3)(2,2,7)GTP-capped mRNAs may be regulated by intracellular redox state.
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA.
Hiroshi
H
Miyoshi
Donard S
DS
Dwyer
Brett D
BD
Keiper
Marzena
M
Jankowska-Anyszka
Edward
E
Darzynkiewicz
Robert E
RE
Rhoads
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D003553:Cystine / Q000737:chemistry; D039561:Eukaryotic Initiation Factor-4E; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D029742:Caenorhabditis elegans Proteins; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D020033:Protein Isoforms; D012315:RNA Caps; D003553:Cystine