An extended RNA binding site for the yeast branch point-binding protein and the role of its zinc knuckle domains in RNA binding.
The highly conserved branch point sequence (BPS) of UACUAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.
Garrey
Stephen M
SM
Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Voelker
Rodger
R
Berglund
J Andrew
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012689:Sequence Homology, Nucleic Acid; D015032:Zinc / Q000737:chemistry*
C409040:MSL5 protein, S cerevisiae; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D015032:Zinc