Diverse role of three tyrosines in binding of the RNA 5' cap to the human nuclear cap binding complex.
The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5' cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants (K(as)) for two selected cap analogues, P(1)-7-methylguanosine-5' P(3)-guanosine-5' triphosphate and 7-methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation-pi stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (DeltaG degrees) with the stacking energy (DeltaE) of the 7-methylguanosine/Y binary and ternary complexes calculated by the Møller-Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the "CBC-to-eukaryotic initiation factor 4E handoff" of mRNA.
Worch
Remigiusz
R
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 93 Zwirki and Wigury St., 02-089 Warszawa, Poland.
Jankowska-Anyszka
Marzena
M
Niedzwiecka
Anna
A
Stepinski
Janusz
J
Mazza
Catherine
C
Darzynkiewicz
Edward
E
Cusack
Stephen
S
Stolarski
Ryszard
R
D019943:Amino Acid Substitution / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D039502:Nuclear Cap-Binding Protein Complex / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D039502:Nuclear Cap-Binding Protein Complex; D012315:RNA Caps; D014443:Tyrosine