Mechanism of dimerization of bicoid mRNA: initiation and stabilization.
Dimerization of bcd mRNA was shown to be important for the formation of ribonucleoprotein particles and their localization in Drosophila embryo. The cis-element responsible for dimerization is localized in a stem-loop domain (domain III) containing two essential complementary 6-nucleotide sequences in a hairpin loop (LIIIb) and an interior loop (LIIIa). Such an RNA element can potentially generate single or double "hand-by-arm" interactions leading to open and closed complexes, respectively. The former retains the possibility of forming multimers, whereas the latter does not. We showed previously that dimerization proceeds through a two-step mechanism, which includes a transition from the reversible initiation complex into a very stable one. Here we have addressed the nature of the initial interactions and the mechanism of transition. We engineered a series of different RNA fragments with the capacity to form defined open dimers, multimers, or closed dimers. We compared their thermodynamic and kinetic behavior and mapped nucleotides involved in intermolecular interactions by enzymatic and chemical footprinting experiments and chemical modification interference. Our results indicate that the initiation step leads to a reversible open dimer, involving a more limited number of intermolecular base pairs than expected. The two loops play distinct roles in this process, and the structure of loop IIIb is more constrained than that of loop IIIa. Thus, loop IIIa appears to be the driving element of the recognition process. The initial open dimer is then converted into a stable closed dimer, possibly through a kinetically controlled mechanism.
Wagner
Céline
C
Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9002 du CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France.
Ehresmann
Chantal
C
Ehresmann
Bernard
B
Brunel
Christine
C
D000818:Animals; D001483:Base Sequence; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics; D015534:Trans-Activators / Q000235:genetics*
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C056478:bcd protein, Drosophila