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Double-stranded RNA homopolymer poly(rC).poly(rG) has been used as a new pH-sensitive drug carrier. The poly(rC).poly(rG) had proton buffering capacity around pH 6, owing to protonation of cytosine, as determined by acid-base titration. By circular dichroism measurement, the protonation caused conformational change of the RNA. The poly(rC).poly(rG) and doxorubicin (Dox), as an anticancer drug, formed the complexes which released the drugs at endosomal pH. The resulting complex exhibited higher anticancer activity than the Dox alone. These results result suggest that the poly(rC).poly(rG) is a promising biopolymer for a new class of pH-sensitive drug carriers.

G-Rich sequences are known to form four-stranded structures that are based on stacks of G-quartets, and sequences with the potential to adopt these structures are common in eukaryotic genomes. However, there are few rules for predicting the relative stability of folded complexes that are adopted by sequences with different-length G-tracts or variable-length linkers between them. We have used thermal melting, circular dichroism, and gel electrophoresis to examine the topology and stability of intramolecular G-quadruplexes that are formed by sequences of the type d(GnT)4 and d(GnT2)4 (n = 3-7) in the presence of varying concentrations of sodium and potassium. In the presence of potassium or sodium, d(GnT)4 sequences form intramolecular parallel complexes with the following order of stability: n = 3 > n = 7 > n = 6 > n = 5 > n = 4. d(G3T)4 is anomalously stable. In contrast, the stability of d(GnT2)4 increases with the length of the G-tract (n = 7 > n = 6 > n = 5 > n = 4 > n = 3). The CD spectra for d(GnT)4 in the presence of potassium exhibit positive peaks around 260 nm, consistent with the formation of parallel topologies. These peaks are retained in sodium-containing buffers, but when n = 4, 5, or 6, CD maxima are observed around 290 nm, suggesting that these sequences [especially d(G5T)4] have some antiparallel characteristics. d(G3T2)4 adopts a parallel conformation in the presence of both sodium and potassium, while all the other d(GnT2)4 complexes exhibit predominantly antiparallel features. The properties of these complexes are also affected by the rate of annealing, and faster rates favor parallel complexes.

The sequence G37pG36 is present in all tRNA species recognized and methylated by the Escherichia coli modification enzyme tRNA (guanosine-1)methyltransferase. We have examined whether this dinucleotide sequence provides the base specific recognition signal for this enzyme and have assessed the role of the remaining tRNA in recognition. E. coli tRNAHis and yeast tRNAAsp were substituted with G at positions 36 and 37 and were found to be excellent substrates for methylation. This suggested that the general tRNA structure can be specifically bound by the enzyme. In addition, heterologous tRNA species including fully modified tRNA1Leu are excellent inhibitors of tRNA1Leu transcript methylation. Analyses of structural variants of yeast tRNAAsp and E. coli tRNA1Leu demonstrate clearly that the core tertiary structures of tRNA are required for recognition and that G37 must be in the correct position in space relative to important contacts elsewhere in the molecule. This latter conclusion was reached because the addition of one to three stacked base pairs in the anticodon stem of tRNA1Leu dramatically alters activity. In this case, the G37 base is rotated away from the correct position in space relative to other tRNA contact sites. The acceptor stem structure is required for optimal activity since deletion of three or five base pairs is detrimental to activity; however, specific base sequence may not be important because (i) the addition of three stacked base pairs of different sequence had little effect on activity and (ii) heterologous tRNAs with little or no sequence homology in the acceptor stem are excellent substrates. Both poly G and GpG are potent and specific inhibitors of enzyme activity and are minimal substrates which can be methylated, forming m1G. Taken together, these studies suggest that 1MGT can bind the general tRNA structure and that the crucial base-pair contacts are G37 and G36.

Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether. We apply this method for the interaction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, and fingers domains. It was proposed that template DNA binds in the cleft. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linked tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal structure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526-5538] we used site-specific psoralen furan-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polymerase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to identify the location of this peptide on the T7 RNA polymerase primary sequence. In the present work we have mapped this peptide on the 3-D structure of T7 RNA polymerase. This peptide maps in the fingers domain of the polymerase. On the basis of a comparison of the map positions of peptides that cross-linked to either promoter DNA or single-stranded oligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-stranded DNA. Whereas the cleft of the polymerase is the seat of double-stranded promoter binding, the fingers domain may be used by the polymerase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alternatively, the single-stranded oligo binding site may be an RNA product-binding site during transcription. The photochemical techniques we have developed [Sastry et al. (1993) Biochemistry 32, 5526-5538; this work] can be applied to other DNA-protein complexes to map DNA-binding domains.

DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli. An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3'-->5' proofreading exonuclease activity resident in the epsilon subunit. Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified epsilon subunit. The steady state kinetic constants are K(m) = 16 +/- 6 microM and kcat = 210 +/- 23 s-1 for degradation of single-stranded DNA by epsilon. These steady state values are in agreement with the rate constants determined for excision of the 3' nucleotide of a dT10 oligomer under pre-steady state conditions. Using a simple two-step model, E + Dn reversible E.Dn-->E + Dn-1, we find K = 12 microM and kf = 280 s-1 for the dT10 substrate. In these experiments, epsilon subunit acts in a distributive manner and product release is not the rate-limiting step. Activity of the epsilon subunit on paired DNA oligonucleotides with zero to three mismatches at the 3' terminus indicates that an additional step is required in the mechanism. In the scheme Dn reversible Dn* + E reversible E.Dn*-->E + Dn-1, the 3' terminus undergoes a conformational change or "melts" before the DNA is a substrate for epsilon subunit. With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA. The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by epsilon subunit is the DNA-melting step.

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant HIV-1 Rev protein was overexpressed in Escherichia coli using translational coupling to the beta-glucuronidase gene and demonstrated to interact with high affinity and specificity with the Rev responsive element (RRE). A complex Rev-dependent binding pattern was observed using the gel shift assay which could be simplified to one or two primary bands in the presence of stoichiometric concentrations of RRE. Competition of these bands with a series of homopolymer RNA species demonstrated that Rev is essentially a poly-G binding protein, although poly-I was also shown to compete for specific RRE binding. The stoichiometry of the Rev-dependent gel shift complexes was determined using 125I-labeled Rev. The stable, lowest mobility complex was determined to possess a ratio of between 7 and 8 Rev molecules per RRE containing RNA fragment while the two fastest migrating complexes contained ratios of one and two Rev molecules per RRE, respectively. Using the Hill equation as a model for cooperative interactions, a Hill coefficient of n(app) = 2 was obtained from fitting of direct nitrocellulose filter binding assays, reflecting cooperatively bound Rev molecules on the RRE under equilibrium binding conditions. An increase in ionic strength from 0.0 to 0.3 M NaCl reduced cooperative Rev binding to the RRE, but specificity of Rev for the RRE relative to antisense RNA was increased 100,000-fold. At molar ratios of Rev to RRE above 2, Rev dissociated from the RRE with a T1/2 of approximately 20-25 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.