Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.

The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template. A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase. A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates. The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined.

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or ‘transcripts’ of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non‐conservative mutations of this tyrosine also reduce discrimination between deoxyribo‐ and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA‐directed DNA polymerase, reverse transcriptase, RNA‐directed RNA polymerase or DNA‐directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.