Steve C. Schultz

Molecular basis of double‐stranded RNA‐protein interactions: structure of a dsRNA‐binding domain complexed with dsRNA

Protein interactions with double‐stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein‐dsDNA interactions, surprisingly little is known about the molecular basis of protein‐dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an ∼70 amino acid motif referred to as the dsRNA‐binding domain (dsRBD). We have determined a 1.9 Å resolution crystal structure of the second dsRBD of Xenopus laevis RNA‐binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A‐form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD‐dsRNA complex and models proposed for polynucleotidyl transferase‐nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.

Oligomeric structures of poliovirus polymerase are important for function

Central to the replication of poliovirus and other positive‐strand RNA viruses is the virally encoded RNA‐dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase–polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase–polymerase interaction. To explore potential functional roles for the structurally observed polymerase–polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross‐linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase–polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase–polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

Dimeric structure of the Oxytricha nova telomere end-binding protein alpha-subunit bound to ssDNA.

Telomeres are the specialized protein–DNA complexes that cap and protect the ends of linear eukaryotic chromosomes. The extreme 3′ end of the telomeric DNA in Oxytricha nova is bound by a two-subunit sequence-specific and 3′ end-specific protein called the telomere end-binding protein (OnTEBP). Here we describe the crystal structure of the α-subunit of OnTEBP in complex with T4G4 single-stranded telomeric DNA. This structure shows an (α–ssDNA)2 homodimer with a large ∼7,000 Å2 protein–protein interface in which the domains of α are rearranged extensively from their positions in the structure of an α–β–ssDNA ternary complex. The (α–ssDNA)2 complex can bind two telomeres on opposite sides of the dimer and, thus, acts as a protein mediator of telomere–telomere associations. The structures of the (α–ssDNA)2 dimer presented here and the previously described α–β–ssDNA complex demonstrate that OnTEBP forms multiple telomeric complexes that potentially mediate the assembly and disassembly of higher order telomeric structures.