Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability.

Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys(2)-His(2) zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d(5)-N-ethylmaleimide (d(5)-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn(6) MTF-zf. A chase with excess H(5)-N-ethylmaleimide (H(5)-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d(5),d(5)-, d(5),H(5)-, and H(5),H(5)-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d(5)-NEM pulse time. This experiment establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 >> F1 > F2 approximately F3 approximately F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that determined for the DNA binding domain of Sp1, Zn(3) Sp1-zf, under identical solution conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-containing oligonucleotide. An identical experiment carried out with Zn(5) MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.

Metal response element (MRE)-binding transcription factor-1 (MTF-1): structure, function, and regulation.

Metal-responsive control of the expression of genes involved in metal metabolism and metal homeostasis allows an organism to tightly regulate the free or bioavailable concentration of beneficial metal ions, such as zinc, copper, and iron, within an acceptable range, while efficiently removing nonbeneficial or toxic metals. Emerging evidence also suggests that metal homeostasis is intimately coupled to the oxidative stress response in many cell types. The expression of genes that encode metallothioneins in all vertebrate cells is strongly induced by potentially toxic concentrations of zinc and cadmium, as well as in response to strong oxidizing agents, including hydrogen peroxide. This induction requires a cis-acting DNA element, termed a metal response element (MRE), and MRE-binding transcription factor-1 (MTF-1), a Cys2-His2 zinc finger protein. This review summarizes recent progress that has been made toward understanding the structure, function, and metalloregulation of mammalian MTF-1.

Conformational heterogeneity in the C-terminal zinc fingers of human MTF-1: an NMR and zinc-binding study.

The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2)-His(2) motifs, each of which was projected to form well-structured betabetaalpha domains upon Zn(II) binding. In this report, the structure and backbone dynamics of a fragment containing the unusual C-terminal fingers F4-F6 has been investigated. (15)N heteronuclear single quantum coherence (HSQC) spectra of uniformly (15)N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn(3) hMTF-zf46 determined by (13)Calpha chemical shift indexing and the magnitude of (3)J(Halpha-HN) clearly reveal that zinc fingers F4 and F6 adopt typical betabetaalpha structures. An analysis of the heteronuclear backbone (15)N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-MTF-zf46 with Zn(II) reveals that the F4 domain binds Zn(II) significantly more tightly than do the other two finger domains. In contrast to fingers F4 and F6, the betabetaalpha fold of finger F5 is unstable and only partially populated at substoichiometric Zn(II); a slight molar excess of zinc results in severe conformational exchange broadening of all F5 NH cross-peaks. Finally, although Cd(II) binds to apo-hMTF-zf46 as revealed by intense S(-)-->Cd(II) absorption, a non-native structure results; addition of stoichiometric Zn(II) to the Cd(II) complex results in quantitative refolding of the betabetaalpha structure in F4 and F6. The functional implications of these results are discussed.

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.

The zinc metalloregulatory protein Synechococcus PCC7942 SmtB binds a single zinc ion per monomer with high affinity in a tetrahedral coordination geometry.

The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).

Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems.

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.

Spectroscopic characterization of Co(II)-, Ni(II)-, and Cd(II)-substituted wild-type and non-native retroviral-type zinc finger peptides.

The nucleocapsid protein (NCP) from Mason-Pfizer monkey virus (MPMV) contains two evolutionary invariant Cys-X2-Cys-X4-His-X4-Cys retroviral-type zinc finger structures, where the Cys and His residues provide ligands to a tetrahedrally coordinated Zn(II) ion. The N-terminal zinc finger (F1) of NCP from MPMV contains an immediately contiguous Cys in the -1 position relative to the start of this conserved motif: Cys-Cys-X2-Cys-X4-His-X4-Cys. Metal complexes of 18-amino acid peptides which model the native zinc finger sequence, SER-Cys-X2-Cys-X4-His-X4-Cys (F1-SC), and non-native Cys-SER-X2-Cys-X4-His-X4-Cys (F1-CS) and SER-SER-X2-Cys-X4-His-X4-Cys (F1-SS) sequences have been spectroscopically characterized and compared to the native two-zinc-finger protein fragment, MPMV NCP 21-80. All Co(II)-substituted peptide complexes adopt tetrahedral ligand geometries and have S- -->Co(II) ligand-to-metal charge-transfer (LMCT) transition intensities consistent with three Co(II)-S bonds for F1-SC and F1-CS. The non-native F1-CS peptide binds Co(II) with KCo= 1.5 x 10(6) M(-1), comparable to that of the native complex, and approximately 100-fold tighter than F1-SS. Like the Co(II) derivative, the absorption spectrum of Ni(II)-substituted NCP 21-80 is most consistent with tetrahedral Ni(II) complexes with multiple thiolate donors. In contrast, Ni(II) complexes of F1-SC and F1-CS exhibit a single absorption band in the 400-550 nm region (epsilon approximately 200-300 M(-1) cm (-1), distinct in the two complexes, assignable to a degenerate d-d transition envelope characteristic of non-native square-planar coordination geometry, and an intense LMCT transition in the UV (epsilon255 approximately 14,000 M(-1) cm(-1)). Cd(II) complexes have intense absorption in the UV (lambda(max)=233nm), with absolute intensities consistent with approximately 5000 M(-1) cm(-1) per Cd(II)-S bond. 113Cd NMR spectroscopy of 113Cd MPMV NCP gives delta=649 ppm, consistent with S3N coordination. Co(II) and Cd(II) complexes of non-native F1-CS peptides are more sensitive to oxidation by O2, relative to F1-SC, suggestive of a higher lability in the non-native chelate. The implications of these findings for the evolutionary conservation of this motif are discussed.

Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting.

Programmed -1 ribosomal frameshifting has become the subject of increasing interest over the last several years, due in part to the ubiquitous nature of this translational recoding mechanism in pathogenic animal and plant viruses. All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a heptanucleotide "slippery sequence" conforming to the general form X XXY YYZ, followed by an RNA structural element, usually an H-type RNA pseudoknot, positioned an optimal number of nucleotides (5 to 9) downstream. The slippery sequence itself promotes a low level ( approximately 1 %) of frameshifting; however, downstream pseudoknots stimulate this process significantly, in some cases up to 30 to 50 %. Although the precise molecular mechanism of stimulation of frameshifting remains poorly understood, significant advances have been made in our knowledge of the three-dimensional structures, thermodynamics of folding, and functional determinants of stimulatory RNA pseudoknots derived from the study of several well-characterized frameshift signals. These studies are summarized here and provide new insights into the structural requirements and mechanism of programmed -1 ribosomal frameshifting.

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus.

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).

Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot.

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.

MRE-Binding transcription factor-1: weak zinc-binding finger domains 5 and 6 modulate the structure, affinity, and specificity of the metal-response element complex.

MRE-binding transcription factor-1 (MTF-1) contains six Cys(2)-His(2) zinc finger sequences, and it has been suggested that the zinc finger domain itself may function as a zinc sensor in zinc-activated expression of metallothioneins (MTs). Previous work has shown that a subset ( approximately 3-4) of the zinc fingers in MTF-zf play a structural role in folding and high-affinity metal-response element (MREd) binding, while one or more other fingers have properties consistent with a metalloregulatory role (weak zinc binding affinity in the absence of DNA). We show here that zinc fingers 5 and 6 correspond to the weak zinc-binding fingers in MTF-zf. Limited trypsinolysis of a Zn(6)-MTF-zf:MREd complex gives rise to a highly protease-resistant core fragment corresponding to amino acids 137-260 or N-terminal zinc fingers 1-4 of MTF-zf. Characterization of a collection of broken-finger (His --> Asn) and missing-finger mutants of MTF-zf reveals that deletion of zinc fingers 5 and 6 to create MTF-zf14 attenuates MREd binding affinity ( approximately 20-fold), while deletion of fingers 4-6 (MTF-zf13) results in a further 20-fold reduction of binding affinity with a nearly complete loss of specificity. Circular dichroism studies reveal that the binding of MTF-zf to the MREd induces a dramatic alteration of the structure of the MREd from a B-form to a double-helical conformation with A-like features. Formation of stoichiometric complexes with MTF-zf14, H279N (Deltazf5) MTF-zf, and MTF-zf13 induces comparatively less A-like structure. Steady-state fluorescence resonance energy transfer (FRET) spectroscopy has been used to globally define the orientation of the multifinger MTF-zf on the MREd. These experiments suggest that fingers 1-4 are oriented on the highly conserved TGCRCnC side of the MREd with fingers 5-6 bound at or near the gGCCc sequence. These findings are consistent with a model in which the N-terminal zinc fingers in MTF-zf are required for high affinity and specific binding to the consensus TGCRCnC core in a way which is subjected to structural and allosteric modulation by the weak zinc-binding C-terminal zinc fingers.

Thermodynamics of stabilization of RNA pseudoknots by cobalt(III) hexaammine.

Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH(3))(3+)(6) have been determined, relative to divalent Mg(2+), for the folded, partially folded (K(f)), and fully unfolded (K(u)) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two-state unfolding transitions. Trivalent Co(NH(3))(3+)(6) was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a K(f) of 5-8 x 10(4) M(-1) in a background of 0.10 M K(+), or 3- to 5-fold larger than the K(f) obtained for two model RNA hairpins and hairpin unfolding intermediates, and approximately 40-50-fold larger than K(f) for Mg(2+). The magnitude of K(f) was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with K(f) reaching 1.2 x 10(5) M(-1) in 50 mM K(+). Two RNA hairpins were found to have affinities for Co(NH(3))(3+)(6) and Ru(NH(3))(3+)(6) of 1-2 x10(4) M(-1), or approximately 15-fold larger than the K(f) of approximately 1000 M(-1) observed for Mg(2+). Additionally, the K(u) of 4,800 M(-1) for the trivalent ligands is approximately 8-fold larger than the K(u) of 600 M(-1) observed for Mg(2+). These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg(2+) and Co(NH(3))(3+)(6) binding of significantly higher affinity than a "duplexlike" delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2.

Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting.

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.

Equilibrium unfolding (folding) pathway of a model H-type pseudoknotted RNA: the role of magnesium ions in stability.

In T2 and related T-even bacteriophages, the upstream autoregulatory mRNA leader sequence of gene 32 folds into a simple tertiary structural motif, a hairpin (H)-type pseudoknot. This pseudoknot is derived from 32 contiguous nucleotides which form two coaxially stacked helical stems that adopt a pseudocontinuous A-form helical structure. These stems are connected by two nonequivalent single-stranded loops. The equilibrium unfolding pathway of a 36-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T2 gene 32 mRNA pseudoknot has been probed as a function of [Mg2+] by analysis of dual optical wavelength, equilibrium thermal melting profiles. A van't Hoff model based on multiple sequential, two-state unfolding transitions has been applied to the resultant data. Compensatory base pair substitutions incorporated into the helical stems have been used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is minimally described by three sequential unfolding transitions. The helix-helix junction region melts first in a low-enthalpy transition, followed by the unfolding of the remainder of helical stem 2, and then, all of stem 1. The total enthalpy of unfolding (folding) at [Mg2+] >/= 1 mM is accounted for by the secondary structure alone, suggesting that, if any non-Watson-Crick or tertiary structure exists in this conformation, it makes little or no enthalpic contribution to the stability of the molecule. Consistent with this, the [Mg2+] dependence of individual unfolding transitions within the pseudoknot is well-described by differential extents of delocalized binding of Mg2+ to folded and unfolded regions of the molecule. At a fixed [Mg2+], the helix junction region and stem 2 sequester more Mg2+ ions than the stem 1 hairpin; a larger fraction of these ions are then released upon unfolding. Two base or base pair substitution mutant RNAs which destabilize the helical junction and/or the base of stem 2 appear to sequester fewer ions, with a correspondingly smaller number of these ions released upon unfolding.

Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1.

MRE-binding transcription factor-1 (MTF-1) activates the expression of metallothionein (MT) genes in mouse and human cells upon binding to one or more tandem metal-response elements (MREs; 5'-ctnTGCRCnCgGCCc) in the MT promoter. MTF-1 contains six Cys2-His2 zinc finger sequences. Previous work suggests that the zinc finger domain itself may function as a zinc sensor in zinc-activated expression of MTs. To obtain molecular insight into MTF-1 function, a recombinant fragment of MTF-1 containing only the zinc finger domain (denoted MTF-zf) has been purified using nondenaturing conditions and characterized with respect to zinc-binding properties, secondary structure, and DNA-binding activity. Different preparations of MTF-zf, following an anaerobic dialysis to quantify Zn(II) and reduced cysteine (by DTNB reactivity) content, reveal Zn(II)/MTF-zf stoichiometries ranging from 3.3 to 5.5 g at Zn(II) and 11-13 reduced thiolates (12 expected). Far-UV CD spectra reveal indistinguishable secondary structural content in all preparations, i.e., enough to fold just three of six zinc fingers of MTF-zf. Removal of additional zinc from MTF-zf gives rise to an insoluble apoprotein. Complex formation between a Zn5.5 MTF-zf and a coumarin-labeled MREd-containing oligonucleotide as monitored by changes in the anisotropy of the coumarin fluorescence gives a Kapp = 3.8 (+/-0.5) x 10(8) M-1 (pH 7.0, 0.20 M NaCl, 25 degreesC). Investigation of the salt type and concentration dependence of Kapp suggests significant contributions from both cation and anion release upon complex formation. Zn5.5 MTF-zf exhibits a large negative heat capacity of complex formation with MREd and can discriminate among DNA duplexes which have mutations deposited on either the TGCRC core or the C-rich side of the MREd. Air oxidation of Zn5.5 MTF-zf results in the reversible conversion of 6 of the 12 Cys thiolates to 3 disulfide bonds; as expected, this has no effect on the secondary structure of MTF-zf, but results in approximately 30-fold reduction in Kapp to approximately 1.2 x 10(7) M-1. In contrast, fully reduced Zn3.5 MTF-zf binds to the MREd with an affinity and [NaCl] dependence largely indistinguishable from those of Zn5.5 MTF-zf. The zinc fingers in MTF-zf are physically and functionally inequivalent. A subset (approximately 3-4) of zinc fingers plays a structural role in folding and high-affinity MREd binding, while one or more additional fingers have properties potentially consistent with a metalloregulatory role.

Non-nearest neighbor effects on the thermodynamics of unfolding of a model mRNA pseudoknot.

The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively. In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wave-length, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions. The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate tm melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2. We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions. A significant increase in the extent of cleavage by RNase T1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3' single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction. In contrast, truncation of the 5' tail has no effect on the stability of the molecule.

Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic complementation groups.

This study examines the contribution of domain II beta of bacteriophage Mu transposase (A protein), a subdomain of the central catalytic domain II, to the transposition reaction. The properties of several point mutations implicate a role for this domain in facilitating metal-assisted assembly of the synaptic complex, as well as in intramolecular DNA strand transfer. Point mutations as well as deletions in domain II beta can be complemented by those in domain II alpha but not those in domain III alpha. Thus, residues within subdomains II alpha and II beta belong to different catalytic complementation groups.

Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site.

Frameshift and readthrough sites within retroviral messenger RNAs are often followed by nucleotide sequences that have the potential to form pseudoknot structures. In the work presented here, NMR methods were used to characterize the base-pairings and structural features of the RNA pseudoknot downstream of the gag-pro frameshift site of simian retrovirus type-1 (SRV-1) and a functional mutant of the SRV-1 pseudoknot. Evidence is presented that these pseudoknots contain two A-form helical stems of six base-pairs each, connected by two loops, in a classic H-type pseudoknot topology. A particularly interesting feature is that the shorter of the two connecting loops, loop 1, consists of only a single adenosine nucleotide that spans the major groove of stem 2. In this respect, the frameshift-associated pseudoknots are structurally similar to the pseudoknot within the gene 32 mRNA of bacteriophage T2, previously characterized by NMR methods. Despite having similar nucleotide sequences, the solvent exchange rates of the imino protons at the junction of the helical stems in the wild-type and mutant frameshifting pseudoknots differ from each other and from the bacteriophage T2 pseudoknot. The implications of this finding are discussed.