Kathleen B. Hall

RNA–protein interactions

Recent discoveries have revealed that there is a myriad of RNAs and associated RNA-binding proteins that spatially and temporally appear in the cells of all organisms. The structures of these RNA-protein complexes are providing valuable insights into the binding modes and functional implications of these interactions. Even the common RNA-binding domains (RBDs) and the double stranded RNA binding motifs (dsRBMs) have been shown to exhibit a plethora of binding modes.

Evidence for Z-form RNA by vacuum UV circular dichroism

Circular dichroism (CD) spectra in the vacuum UV region for different conformations of poly d(G-C) X poly d(G-C) and poly r(G-C) X poly r(G-C) are very characteristic. The CD of the RNA in the A-form (6 M NaClO4 and 22 degrees C) is very similar to that of the DNA in 80% alcohol where it is believed to be in the A-form. With the exception of the longest wavelength transition, the CD of the RNA in 6 M NaClO4 at 46 degrees C is similar to the CD of the DNA under conditions where it is believed to be in the Z-form (2 M NaClO4). This substantiates that poly r(G-C) X poly r(G-C) assumes a left-handed Z-conformation in 6 M NaClO4 above 35 degrees C. CD spectra for the left-handed Z-forms of both the RNA and DNA are characterized by an intense negative peak at 190-195 nm, a crossover at about 184 nm, and an intense positive peak below 180 nm. The right-handed A- and B-forms of RNA and DNA all have an intense positive peak in their CD spectra near 186 nm. The large difference in CD in the range 185-195 nm for right- and left-handed conformations of nucleic acids can be used to identify the sense of helix winding.

An RBD that does not bind RNA: NMR secondary structure determination and biochemical properties of the C-terminal RNA binding domain from the human U1A protein*

We have obtained backbone 1H, 15N, and 13C assignments and determined the secondary structure and folding topology of the C-terminal RNA-binding domain (RBD) of the human U1A protein. The secondary structure derived from NOE data is in excellent agreement with the predicted structure from the 1H and 13C chemical shift indices. This 88 amino acid domain exhibits a beta alpha beta-beta alpha beta folding pattern, with conserved RNP1 and RNP2 sequences located in two adjacent strands of a four-strand antiparallel beta-sheet. This global folding pattern is typical of this class of RNA binding proteins. Although this domain contains residues that are conserved in all RBDs, its RNA binding properties are very unusual. RNA binding studies show that this domain does not bind U1, U2 or U5 snRNA, an RNA hairpin, rA16, rU16, rC16 or rA3U3GUA4, nor does it show significant association to populations of random sequence RNAs.

RNA in MOTION

Although RNA duplex regions are highly structured and inflexible, other elements of an RNA molecule are capable of dynamic motions. These flexible regions are the sites of interactions with small molecules, proteins, and other RNAs, yet there are few descriptions of these regions that include the timescale and amplitude of their motions. No one technique is sufficient to accurately describe these motions, but the combination of in vitro methods, particularly NMR relaxation methods, and more robust in silico methods, is beginning to yield the type of data that can be used to understand RNA function. Very few RNAs have been described by both techniques, and here one such RNA and one RNA:protein complex are reviewed.

A functional role for correlated motion in the N-terminal RNA-binding domain of human U1A protein.

The N-terminal RNA-binding domain of the human U1A protein (RBD1) undergoes local conformational changes upon binding to its target RNA. Here, the wild-type RBD1 and two mutants are examined with molecular dynamics simulations that are analyzed using the reorientational eigenmode dynamics (RED) formalism. The results reveal changes in the magnitude and extent of coupled intra-domain motions resulting from single amino acid substitutions. Interpretation of the novel RED results and corresponding NMR relaxation data suggests that the loss of collective motions in the mutants could account for their weak RNA-binding.

2-Aminopurine as a Probe of RNA Conformational Transitions

2-aminopurine (2AP) is a fluorescent nucleobase that provides the means to probe structure and dynamics of RNA molecules. Because 2AP can base pair with Uridine, it can replace normal A:U pairs without substantial deformation of duplexes. It is best used as a probe of ostensibly single-stranded regions: its fluorescence intensity reports on base stacking and its fluorescence decay lifetimes report on its conformational dynamics. Three examples of its use are described here, illustrating how 2AP fluorescence has been used to probe RNA folding and hairpin loop dynamics.

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics.

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.

Monte Carlo applications to thermal and chemical denaturation experiments of nucleic acids and proteins.

Publisher Summary This chapter describes the Monte Carlo applications to thermal and chemical denaturation experiments of nucleic acids and proteins. Information about the states of systems must often be extracted indirectly, from the measurements of properties, considered characteristic of these states. However, even for systems, with stable, well-defined states, the ability to determine the true value of any property is complicated by errors in the measurement process. Statistical fluctuations due to processes on the molecular scale may further obfuscate attempts to determine the most likely (probable) value of the property under consideration. As an example of this process, the chapter considers the measurement of the absorbance of a ribonucleic acid (RNA) solution under constant solution conditions (constant temperature, pH, salt concentration, and nucleic acid concentration). Nucleic acid structural transitions are commonly monitored, by absorbance measurements, in the ultraviolet region of the spectrum, from which the changes in species fractions can be extracted and enthalpy, entropy, and free-energy changes of unfolding calculated.

[12] Thermodynamics and mutations in RNA—Protein interactions

Publisher Summary This chapter discusses the thermodynamics and mutations in RNA-Protein interactions. The association between an RNA and a protein can be described by defining the local interactions between nucleotides and amino acids and by determining the energetics of the association. The local interactions show how the specificity of the association is conferred; the energetics will provide the assembly parameters that encompass both the individual interactions and their interdependence. To predict the properties of an RNA-protein interaction, it is necessary to know how the specificity and affinity of the interaction are controlled. The details of the association include how the RNA phosphate backbone is used in electrostatic interactions, where hydrogen bonds are formed between RNA and protein, if the two molecules associate to form a hydrophobic core of aromatic amino acids and nucleotides, where water molecules and counterions are used in the interaction, and if, in order to form these interactions, there is any conformational rearrangement of RNA or protein. While the thermodynamic parameters of the interaction certainly not provide all these details, they can suggest which features are likely to be important for the interaction, and provide a framework in which to construct an accurate model of the complex. One simple approach to uncover the interactions and energetics of RNA-protein complexes is to make a mutation in the RNA sequence and then measure the affinity of the protein for this RNA variant.

Interactions between PTB RRMs induce slow motions and increase RNA binding affinity.

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. (15)N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.