Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction

Abstract

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.

Transition state for protein–DNA recognition

“…These behaviors may be due to different biological requirements for fast binding and kinetic stability of each complex. Our results contribute to the search for a consensus view of sequence readout in the kinetics of protein binding to double-and single-stranded nucleic acids (40,41), equivalent to recent progress on the understanding of proteinprotein association (35,42).…”

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

“…Our data suggest that its role in positioning the loop is more important than the contacts it makes to Arg52. We and others (Tang and Nilsson 1999;Law et al 2005) had previously proposed a key role for Arg52 in initiating close range interactions between protein and RNA. The modest effect of closing base-pair mutations suggests that the electrostatic role of Arg52, its interaction with A1, and its positioning of the neighboring amino acids Arg47, Ser48, and Gln54 (Law et al 2005) are more important than its interaction with G11 (Fig.…”

“…1B) interact with both amide groups (Nz1 and 2) of Arg52 while the phosphate group of G11 interacts with Leu49. Arg52 also forms a hydrogen bond with A1 in the RNA loop (via Nz1), a step thought to occur early in complex formation (Oubridge et al 1994;Tang and Nilsson 1999;Law et al 2005). These interactions may promote a series of further contacts that lock U1A and U1hpII together.…”

“…These interactions may promote a series of further contacts that lock U1A and U1hpII together. Thus, the stem and the closing base pair may play a role in initiating close-range interactions (Law et al 2005). In addition, the stem also interacts electrostatically with U1A.…”

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

“…The complex between the spliceosomal protein U1A and its target on the U1 small nuclear RNA has served as a model system for many studies. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In this complex, the N-terminal RNA recognition motif of U1A interacts in a sequence-specific manner with stem/loop II (U1SLII) of the U1 small nuclear RNA ( Figure 1). Both experimental and computational studies have probed determinants of the binding affinity, and a number of experimental studies 1,7,9,10,[13][14][15] have been carried out to dissect the binding rate.…”