Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.
The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.
Carberry
S E
SE
Department of Chemistry, Hunter College of the City University of New York 10021-5024.
Friedland
D E
DE
Rhoads
R E
RE
Goss
D J
DJ
D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry; D039561:Eukaryotic Initiation Factor-4E; D005453:Fluorescence; D005914:Globins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry*; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D012315:RNA Caps / Q000737:chemistry*; D011817:Rabbits; D013329:Structure-Activity Relationship; D014764:Viral Proteins
D039561:Eukaryotic Initiation Factor-4E; D009843:Oligoribonucleotides; D010448:Peptide Initiation Factors; D012315:RNA Caps; D014764:Viral Proteins; D005914:Globins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases