Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme

The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechanism. The 8-17 bound its substrate with a rate constant ~10-fold lower than those typical for the annealing of short complementary oligonucleotides. The observed free energy of substrate binding indicates that an energetic penalty near to +7 kcal/mol is attributable to the deoxyribozyme core. Substrate cleavage required divalent metal ion cofactors, and the dependence of activity on the concentration of Mg²⁺, Ca²⁺ or Mn²⁺ suggests the occurrence of a single, low-specificity binding site for activating ions. The efficiency of activation correlated with the Lewis acidity of the ion cofactor, compatible with a metal-assisted deprotonation of the reactive 2′-hydroxyl group. However, alternative roles of the metal ions cannot be excluded, because those ions that are stronger Lewis acids are also capable of forming stronger interactions with ligands such as the phosphate oxygens. The apparent enthalpy of activation for the 8-17 reaction was close to the values observed for hydroxide-catalyzed and hammerhead ribozyme-catalyzed RNA cleavage.     

In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the ‘8–17’ deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can be either ribo- or deoxyribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn²⁺, the cleavage rate is 1.35 min^–1 at pH 6.0; based on pH profile this rate is estimated to be at least ~30 times faster at pH 7.5, where most assays of Mg²⁺-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn²⁺ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg²⁺-dependent nucleic acid enzymes under similar conditions.     

Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme

We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5′-phosphate and 3′-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs=2.7 h(?1) and rate enhancement of 10(12) over the uncatalyzed P?O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson?Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson?Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson?Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its “recognition site” from ATGT (as for 10MD5) to ATGTT or larger, i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.     

ERK activity and G1 phase progression: identifying dispensable versus essential activities and primary versus secondary targets

The ERK subfamily of MAP kinases is a critical regulator of S phase entry. ERK activity regulates the induction of cyclin D1, and a sustained ERK signal is thought to be required for this effect, at least in fibroblasts. We now show that early G1 phase ERK activity is dispensable for the induction of cyclin D1 and that the critical ERK signaling period is restricted to 3-6 h after mitogenic stimulation of quiescent fibroblasts. Similarly, early G1 phase ERK activity is dispensable for entry into S phase. Moreover, if cyclin D1 is expressed ectopically, ERK activity becomes dispensable throughout the G1 phase. In addition to its effect on cyclin D1, ERK activity is thought to contribute to the down-regulation of p27kip1. We found that this effect is restricted to late G1/S phase. Mechanistic analysis showed that the ERK effect on p27kip1 is mediated by Skp2 and is secondary to its effect on cyclin D1. Our results emphasize the importance of mid-G1 phase ERK activity and resolve primary versus secondary ERK targets within the G1 phase cyclin-dependent kinases.     

An analysis of intron positions in relation to nucleotides, amino acids, and protein secondary structure

We present an analysis of intron positions in relation to nucleotides, amino acid residues, and protein secondary structure. Previous work has shown that intron sites in proteins are not randomly distributed with respect to secondary structures. Here we show that this preference can be almost totally explained by the nucleotide bias of splice site machinery, and may well not relate to protein stability or conformation at all. Each intron phase is preferentially associated with its own set of residues: phase 0 introns with lysine, glutamine, and glutamic acid before the intron, and valine after; phase 1 introns with glycine, alanine, valine, aspartic acid, and glutamic acid; and phase 2 introns with arginine, serine, lysine, and tryptophan. These preferences can be explained principally on the basis of nucleotide bias at intron locations, which is in accordance with previous literature. Although this work does not prove that introns are inserted into genomes at specific proto-splice sites, it shows that the nucleotide bias surrounding introns, however it originally occurred, explains the observed correlations between introns and protein secondary structure.     

A reinvestigation of the secondary structure of functionally active vSGLT, the vibrio sodium/galactose cotransporter

The bacterial Na(+)/galactose cotransporter vSGLT of Vibrio parahaemolyticus is a member of the sodium:solute symporter family (SSS). Previous studies using electron microscopy have shown that vSGLT is a monomeric protein. Computational and experimental topological analyses have consistently indicated that this protein possesses 14 transmembrane alpha-helices. Our previous study using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to quantitate secondary structure content had indicated, in contrast, an alpha-helical content of only 35%, too little to be consistent with the 14-span model [le Coutre, J., et al. (2002) Biochemistry 41, 8082-6]. ATR-FTIR had also indicated that upon binding of Na(+) and d-galactose, the alpha-helical content increased to 53%. Here we revisit the vSGLT secondary structural distribution using an alternative approach, ultraviolet circular dichroism spectropolarimetry (CD), which is highly accurate in determining the alpha-helical content of a protein in solution. CD spectra were obtained from actively functional, soluble vSGLT and, as an internal check, from a fusion protein of vSGLT and the beta-barrel green fluorescent protein (GFP). Far-UV CD of vSGLT indicates a predominating 85% alpha-helical content, and an absence of beta-strands. Far-UV CD of the vSGLT-GFP fusion corroborates this profile, indicating an equivalent alpha-helical content, and a beta-strand content consistent with the GFP contribution. No detectable substrate-induced macroscopic changes in secondary structure are apparent in the far UV. In the near UV, increases in positive CD intensity occur in a stepwise manner with added substrates, implying changing environments of aromatic amino acid residues. CD thus confirms the current 14-transmembrane span model of vSGLT and reveals distinct substrate-induced conformational changes. The high percentage of alpha-helical structure found requires, when considered in the context of membrane topology, that nearly a third of the total alpha-helical fraction lies in extramembrane domains, which distinguishes this cotransporter from the unrelated lactose and glycerol 3-phosphate transporters.     

Crystal structure of Dps-1, a functionally distinct Dps protein from Deinococcus radiodurans

DNA protection during starvation (Dps) proteins play an important role in protecting cellular macromolecules from damage by reactive oxygen species (ROS). Unlike most orthologs that protect DNA by a combination of DNA binding and prevention of hydroxyl radical formation by ferroxidation and sequestration of iron, Dps-1 from the radiation-resistant Deinococcus radiodurans fails to protect DNA from hydroxyl radical-mediated cleavage through a mechanism inferred to involve continuous release of iron from the protein core. To address the structural basis for this unusual release of Fe(2+), the crystal structure of D. radiodurans Dps-1 was determined to 2.0 Angstroms resolution. Two of four strong anomalous signals per protein subunit correspond to metal-binding sites within an iron-uptake channel and a ferroxidase site, common features related to the canonical functions of Dps homologs. Similar to Lactobacillus lactis Dps, a metal-binding site is found at the N-terminal region. Unlike other metal sites, this site is located at the base of an N-terminal coil on the outer surface of the dodecameric protein sphere and does not involve symmetric association of protein subunits. Intriguingly, a unique channel-like structure is seen featuring a fourth metal coordination site that results from 3-fold symmetrical association of protein subunits through alpha2 helices. The presence of this metal-binding site suggests that it may define an iron-exit channel responsible for the continuous release of iron from the protein core. This interpretation is supported by substitution of residues involved in this ion coordination and the observation that the resultant mutant protein exhibits significantly attenuated iron release. Therefore, we propose that D. radiodurans Dps-1 has a distinct iron-exit channel.     

Towards elucidation of the mechanism of UV1C, a deoxyribozyme with photolyase activity

Among the unexpected chemistries that can be catalyzed by nucleic acid enzymes is photochemistry. We have reported the in vitro selection of a small, cofactor-independent deoxyribozyme, UV1C, capable of repairing thymine dimers in a DNA substrate, most optimally with light at a wavelength of >300 nm. We hypothesized that a guanine quadruplex functioned both as a light antenna and an electron source for the repair of the substrate within the enzyme-substrate complex. Here, we report structural and mechanistic investigations of that hypothesis. Contact-crosslinking and guanosine to inosine mutational studies reveal that the thymine dimer and the guanine quadruplex are positioned close to each other in the deoxyribozyme-substrate complex, and permit us to refine the structure and topology of the folded deoxyribozyme. In exploring the substrate utilization capabilities of UV1C, we find it to be able to repair uracil dimers as well as thymine dimers, as long as they are present in an overall deoxyribonucleotide milieu. Some surprising similarities with bacterial CPD photolyase enzymes are noted.     

Fluorescence studies of the interaction between 1,N6-ethenoadenosine monophosphate and nucleotides.

AMP, GMP, TMP and CMP quench the fluorescence of 1,N6-ethenoadenosine monophosphate (epsilon-AMP). The fluorescence spectrum of epsilon-AMP-nucleotide system is identical with that of epsilon-AMP itself, and the fluorescence decay kinetics follow a single-exponential decay law. The dependence of fluorescence yields and lifetimes upon the concentration of nucleotides shows that the fluorescence of epsilon-AMP is principally quenched in a dynamic process by AMP, TMP and CMP, while it is quenched in both dynamic and static processes by GMP. The quenching constants increase in the following order: GMP greater than AMP greater than TMP greater than CMP.     

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6

Summary Essentially complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [¹⁵N]-and [¹⁵N, ¹³C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of ¹³C-chemical shift, amide hydrogen-bond exchange, and ¹⁵N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five -helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.     

Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin

The partly folded state of apomyoglobin at pH 4 represents an excellent model for an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, (1)H-(1)H NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T(1) and T(2) and ?(1)H?-(15)N NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the (13)C(alpha), (13)CO, (1)H(alpha), and (13)C(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of (1)H-(1)H NOEs. Hydrogen bonding in the structured regions is predominantly native-like according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a native-like structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.     

Approach to identifying the functionally important segments of RNA, based on complementation-addressed modification

An approach based on complementation-addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5'-P and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect, whereas alteration of hairpin IIIe substantially reduced the binding. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play a critical role in the binding of HCV IRES with the 40S subunit.     

Expression, purification, and secondary structure characterization of recombinant KCTD1

Potassium channel tetramerization domain containing 1 (KCTD1) contains a BTB domain, which can facilitate protein-protein interactions that may be involved in the regulation of signaling pathways. Here we describe an expression and purification system that can provide a significant amount of recombinant KCTD1 from Escherichia coli. The cDNA encoding human KCTD1 was amplified and cloned into the expression vector pET-30a(+). The recombinant protein was expressed in E. coli BL21(DE3) cells and subsequently purified using affinity chromatography. To confirm that KCTD1 was correctly expressed and folded, the molecular weight and conformation were analyzed using mass spectroscopy, Western blot, and circular dichroism. Optimizing KCTD1 expression and investigating its secondary structure will provide valuable information for future structural and functional studies of KCTD1 and KCTD family proteins.     

Improved methods for determining the concentration of 6-thioguanine nucleotides and 6-methylmercaptopurine nucleotides in blood

The conversion of the cytotoxic and immunosuppressive 6-mercaptopurine (6MP) to the active 6-thioguanine nucleotides (6TGN) is necessary for clinical efficacy of 6MP and its prodrug azathioprine. Another metabolite, 6-methylmercaptopurine nucleotide (6MMPN), is formed via a competing pathway by thiopurine methyl transferase. The concentrations of 6TGN and 6MMPN are measured in washed erythrocytes as a surrogate to the intracellular levels of these metabolites in the target tissues. Analysis of 6TGN and 6MMPN in multi-center clinical studies is more complicated because of the requirement to wash erythrocytes. In this investigation, we found no differences in the concentrations of 6TGN and 6MMPN in blood versus washed erythrocytes in samples obtained from patients taking therapeutic doses of oral 6MP or azathioprine for inflammatory bowel disease. We concluded that whole blood could be used for the analysis of these analytes, thus saving sample preparation time. We also found that the erythrocyte 6TGN concentration in blood at ambient temperature declined 2–4% per day, a loss that can be avoided by shipping blood samples frozen. The loss of 6TGN in blood stored at approximately −80°C was 1% after 1 week and 12% after 24 weeks, indicating the analyte was moderately stable. 6MMPN in blood did not significantly change after 24 weeks of storage at approximately −80°C. In addition, the sensitivity of the 6TGN assay was improved by modifying the HPLC conditions, which made the method more suitable for quantifying low levels of 6TGN in human intestinal biopsy samples and blood.     

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.     

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase. The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.     

Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.