Locked tether formation by cooperative folding of Rna14p monkeytail and Rna15p hinge domains in the yeast CF IA complex

The removal of the 3' region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.     

ATP‐dependent DNA binding,unwinding, and resection by the Mre11/Rad50 complex

ATP‐dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double‐strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MR‐ATPγS‐DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS‐bound Rad50 nucleotide‐binding domains. Duplex DNA cannot access the Mre11 active site in the ATP‐free full‐length MR complex. ATP hydrolysis drives rotation of the nucleotide‐binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis‐driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.     

Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation

In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.     

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.     

ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli.

Clp protease is a high relative molecular mass, ATP-dependent protease found in the cytoplasm of Escherichia coli. Clp protease is composed of two protein components, Clp A, which has ATPase activity, and Clp P, which has the proteolytic active site and is activated by Clp A in the presence of ATP. Clp P subunits (Mr = 21,500) are arranged in two hexagonal rings directly superimposed on each other, and under low salt conditions two dodecamers associate to form a particle with Mr approximately 440,000. Clp A (subunit Mr = 83,000) and Clp P do not associate in the absence of nucleotide, but Clp A with ATP bound associates with Clp P to form an active proteolytic complex with Mr approximately 700,000. Although adenosine 5'-[beta gamma-imido]triphosphate (AMPPNP) weakly promotes association between Clp A and Clp P, non-hydrolysable analogues of ATP do not activate proteolysis, indicating that association between the components is not sufficient to allow proteolysis. Association between Clp A and Clp P does not alter the basal ATPase activity of Clp A, but addition of protein substrates is accompanied by an increase in ATP hydrolysis by Clp A. Chemically-inactivated Clp P or inactive mutants of Clp P also associate with Clp A, but no increase in the ATPase activity of Clp A is observed, either in the presence or absence of protein substrates, when Clp P is inactive. Thus the increased ATP hydrolysis is dependent on active proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic site modifications of TAP1 and TAP2 and their functional consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.     

Crystal structure of the Rna14-Rna15 complex

A large protein machinery is required for 3'-end processing of mRNA precursors in eukaryotes. Cleavage factor IA (CF IA), a complex in the 3'-end processing machinery in yeast, contains four subunits, Rna14, Rna15, Clp1, and Pcf11. Rna14 has a HAT (half a TPR) domain at the N terminus and a region at the C terminus that mediates interactions with Rna15. Rna15 contains a RNA recognition module (RRM) at the N terminus, followed by a hinge region. These two proteins are homologs of CstF-77 and CstF-64 in the cleavage stimulation factor (CstF) of the mammalian 3'-end processing machinery. We report the first crystal structure of Rna14 in complex with the hinge region of Rna15, and the structures of the HAT domain of Rna14 alone in two different crystal forms. The complex of the C-terminal region of Rna14 with the hinge region of Rna15 does not have strong interactions with the HAT domain of Rna14, and this complex is likely to function independently of the HAT domain. Like CstF-77, the HAT domain of Rna14 is also a tightly associated dimer with a highly elongated shape. However, there are large variations in the organization of this dimer among the Rna14 structures, and there are also significant structural differences to CstF-77. These observations suggest that the HAT domain and especially its dimer may have some inherent conformational variability.     

Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator

The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.     

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.     

Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor

Terminase enzymes are responsible for "packaging" of viral DNA into a preformed procapsid. Bacteriophage lambda terminase is composed of two subunits, gpA and gpNu1, in a gpA(1).gpNu1(2) holoenzyme complex. The larger gpA subunit is responsible for preparation of viral DNA for packaging, and is central to the packaging motor complex. The smaller gpNu1 subunit is required for site-specific assembly of the packaging motor on viral DNA. Terminase assembly at the packaging initiation site is regulated by ATP binding and hydrolysis at the gpNu1 subunit. Characterization of the catalytic and structural interactions between the DNA and nucleotide binding sites of gpNu1 is thus central to our understanding of the packaging motor at the molecular level. The high-resolution structure of the DNA binding domain of gpNu1 (gpNu1-DBD) was recently determined in our lab [de Beer, T., et al. (2002) Mol. Cell 9, 981-991]. The structure reveals the presence of a winged-helix-turn-helix DNA binding motif, but the location of the ATPase catalytic site in gpNu1 remains unknown. In this work, nucleotide binding to the gpNu1-DBD was probed using acrylamide fluorescence quenching and fluorescence-monitored ligand binding studies. The data indicate that the minimal DBD dimer binds both ATP and ADP at two equivalent but highly cooperative binding sites. The data further suggest that ATP and ADP induce distinct conformations of the dimer but do not affect DNA binding affinity. The implications of these results with respect to the assembly and function of a terminase DNA-packaging motor are discussed.     

An interaction between the Walker A and D-loop motifs is critical to ATP hydrolysis and cooperativity in bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.