Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.     

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.     

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps.

Disulfide crosslinking via thiol-disulfide interchange was applied to quantitate the relative flexibility contributed by nicks and single-stranded gaps in an RNA structure. An RNA duplex comprised of three strands was constructed containing the disulfide crosslink precursors 1 and 2 at opposite ends of the duplex on opposite strands. The third strand was of varying length to yield a nick or single-stranded gaps of 1, 2, or 3 nt. Crosslinking rates Indicated relative flexibilities of the resulting two-helix junctions. Crosslinking in the nicked duplex occurred two orders of magnitude slower than in a duplex containing a 3-nt gap. Rates of crosslinking in duplexes with 3-and 2-nt gaps showed only modest dependence on the gap sequence. Many natural RNAs, including ribozymes, contain two-helix junctions related to the model system described here. The data suggest that two-helix junctions containing a nick in one strand will retain substantial rigidity, whereas one or more single-stranded nucleotides at a two-helix junction allow significant flexibility.     

Properties of cloned and expressed human RNase H1.

We have characterized cloned His-tag human RNase H1. The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH. The optimum conditions for catalysis consisted of 1 mM Mg(2+) and pH 7-8. In the presence of Mg(2+), Mn(2+) was inhibitory. Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1. The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5'-phosphate and 3'-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2' modifications at the cleavage site. Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10-20-fold greater affinity than that observed for E. coli RNase H1. The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex. Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5'-RNA-3'-DNA terminus of the duplex. Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide. The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity. The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs.     

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA

The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes. We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein. A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E. coli. Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (approximately 4 M), exhibiting a sharp transition. Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence. The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

RNA binding specificity of Unr, a protein with five cold shock domains

The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD). The capacity of Unr to interact in vitro with RNA and its intracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mRNA targets, we investigated the RNA-binding specificity of Unr by an in vitro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characterized by a conserved core motif AAGUA/G or AACG downstream of a purine stretch. These consensus sequences are 11-14 nt long and appear unstructured. RNAs containing a consensus sequence were bound specifically by Unr with an apparent dissociation constant of 1 x 10(-8) M and both elements, the 5' purine stretch and the core motif, were shown to contribute to the high affinity. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity. Two combinations of CSDs, CSD1-2-3 and CSD1*2-3-4-5 were sufficient to achieve the high affinity of Unr, indicating some redundancy between the CSDs of Unr for RNA recognition. The SELEX-generated consensus motifs for Unr differ from the AACAUC motif selected by the Xenopus Y-box factor FRGY2, indicating that a diversity of RNA sequences could be recognized by CSD-containing proteins.     

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.     

Poly-2'-DNP-RNAs with enhanced efficacy for inhibiting cancer cell growth

It is often believed that small interfering RNA (siRNA) is at least 10-fold more effective than the single-stranded antisense oligonucleotide for silencing the same target gene in the same cells. In view of the recent discovery that the RNA-induced silencing complex (RISC) contains only a single-stranded RNA (ssRNA) molecule and can be reconstituted using single-stranded antisense RNA, such a large difference in efficacy seems puzzling. One possible reason is that hybridization protects siRNA from hydrolysis by endogenous RNase activity until it is incorporated in the RISC, whereas ssRNA is rapidly hydrolyzed. Because the single-stranded poly-2'-O-(2,4-dinitrophenyl)-RNA (DNP-ssRNA) is both RNase resistant and membrane permeable, we synthesized homologous native siRNAs, DNP-siRNAs, native ssRNAs, and DNP-ssRNAs and made a comparative study of their efficacies for inhibiting the growth of two cancer cell lines with different overexpressed target genes under equivalent experimental conditions. It was found that the efficacy of antisense DNP-ssRNA is higher than that of the corresponding siRNA and that the efficacy of native siRNA for inhibiting cell growth can also be enhanced from 2-fold to 6-fold by replacing the native strands of RNA in siRNA with homologous DNP-RNA. Thermal denaturation data show that the hybridization affinity of the DNP-RNA/RNA duplex is higher than that of the native RNA/RNA duplex. Western blotting analysis of A549 cells treated with antisense DNP-ssRNAs containing single mismatching bases shows that the gene silencing by antisense DNP-ssRNA is as sequence specific as that by siRNA. The observed large enhancement of inhibition efficacy of native RNAs by DNP derivatization should be advantageous for both gene silencing studies and therapeutic applications.     

Recognition of double-stranded RNA by guanidine-modified peptide nucleic acids

Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Herein, we tested if the molecular recognition of RNA could be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from d-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.     

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA.

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 base pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabilize the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7-10-fold lower affinity than to CCCTT-containing DNA.