Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus.

Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs.

RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear.

Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities.

DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition.

DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods.

The RING domain of the scaffold protein Ste5 adopts a molten globular character with high thermal and chemical stability.

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the β and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M).

The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA.

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes.

Ligand binding studied by 2D IR spectroscopy using the azidohomoalanine label.

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain.