TY - JOUR
T1 - Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction
AU - Law, Michael J.
AU - Chambers, Eric J.
AU - Katsamba, Phinikoula S.
AU - Haworth, Ian S.
AU - Laird-Offringa, Ite A.
N1 - Funding Information:
We thank members of the Laird-Offringa lab for helpful criticism, and are grateful to Raveendra Dayam, John Barnes and Roland Beckmann for their kind assistance. This work was supported by National Science Foundation Grant MCB-0131782 (to I.A.L.-O.). Funding to pay the Open Access publication charges for this article was provided by National Science Foundation Grant MCB-0131782 to I.A.L.-O.
PY - 2005
Y1 - 2005
N2 - The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem-loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.
AB - The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem-loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.
UR - http://www.scopus.com/inward/record.url?scp=20144377893&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=20144377893&partnerID=8YFLogxK
U2 - 10.1093/nar/gki602
DO - 10.1093/nar/gki602
M3 - Article
C2 - 15914668
AN - SCOPUS:20144377893
SN - 0305-1048
VL - 33
SP - 2917
EP - 2928
JO - Nucleic acids research
JF - Nucleic acids research
IS - 9
ER -