The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional \"tertiary\" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch\'s structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

Kink turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G·A and A·G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G·A and A·G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the presence of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties and the structure adopted on local sequence.

Natural hammerhead ribozymes (HHRz) feature tertiary interactions between hairpin loops or bulges in two of three helices that surround the catalytic core of conserved nucleotides. Their conservation was originally established on minimal versions lacking the tertiary interactions. While those sequence requirements in general also apply to natural versions, we show here differences for the HHRz cleavage site N17. A guanosine at this position strongly impairs cleavage activity in minimal versions, whereas we observe for the G17 variants of four tertiary stabilized HHRz significant cleavage and ligation activity in vitro. Kinetic analyses of these variants revealed a reduced rate and extent of cleavage, compared with wild-type sequences, while variants with distorted tertiary interactions cleaved at a reduced rate, but to the same extent. Contrary to this, G17 variants exhibit similar in vitro ligation activity as compared with the respective wild-type motif. To also address the catalytic performance of these motifs in vivo, we have inserted HHRz cassettes in the lacZ gene and tested this β-galactosidase reporter in Dictyostelium discoideum. In colorimetric assays, we observe differences in the enzymatic activity of β-galactosidase, which correlate well with the activity of the different HHRz variants in vitro and which can be unambiguously attributed to ribozyme cleavage by primer extension analysis.