DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage.

The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb.     

Specificity of the hairpin ribozyme. Sequence requirements surrounding the cleavage site.

Substrate sequence requirements of the hairpin ribozyme have been partially defined by both mutational and in vitro selection experiments. It was considered that the best targets were those that included the N downward arrowGUC sequence surrounding the cleavage site. In contrast to previous studies that failed to evaluate all possible combinations of these nucleotides, we have performed an exhaustive analysis of the cleavage of 64 substrate variants. They represent all possible sequence combinations of the J2/1 nucleotides except the well established G(+1). No cleavage was observed with 24 sequences. C(+2) variants showed little or no cleavage, whereas U(+2) substrates were all cleavable. The maximal cleavage rate was obtained with the AGUC substrate. Cleavage rates of sequences HGUC (H = A, C, or U), GGUN, GGGR (R = A or G), AGUU, and UGUA were up to 5 times lower than the AGUC one. This shows that other sequences besides NGUC could also be considered as good targets. A second group of sequences WGGG (W = A or U), UGUK (K = G or U), MGAG (M = A or C), AGUA, and UGGA were cleaved between 6 and 10 times less efficiently. Furthermore, the UGCU sequence of a noncleavable viral target was mutated to AGUC resulting in a proficiently cleavable substrate by its cognate hairpin ribozyme. This indicates that our conclusions may be extrapolated to other hairpin ribozymes with different specificity.     

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design of hairpin ribozyme variants with improved activity for poorly processed substrates

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y?2 N?1 *G+1 U+2 Y+3 B+?, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U?2 G?1 *G+1 U+2 A+3 A+? sites. Substrates with G?1 *G+1 U+2 A+3 sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+3 and A+? are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G? →U, owing to restoration of a Watson-Crick base pair in helix 1. In this ribozyme-substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A? with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A?, C? and G?. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.     

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.     

Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements.

Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.     

The nucleotide sequence ofBeneckea harveyi 5S rRNA

Summary The complete sequence of the 5S rRNA from the bioluminescent bacterium,Beneckea harveyi has been determined to be p U G C U U G G C G C C A U A G C G A U U-G G A C C C A C U G A (U) C U U C A U U C C-G A A C C A G A A G U G A A C G A A U U A-G G C C G A U G G U G U G U G G G G C U-C C C C A U G U A G A G U A G G A A U C G-C C A G G U (U)_OH.Two sites of sensitivity to ribonuclease T₂ cleavage were identified; at A₄₁ and either A₅₄ or A₅₅. Comparison with existing sequence information fromEscherichia coli andPhotobacterium phosphoreum clarifies the amount of diversity among the bioluminescent bacteria and provides further insight into their phylogenetic position. Sequence heterogeneities were encountered and the importance of these in interpreting 5S rRNA data is discussed.     

ACA‐specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF‐ec) cleaves RNA at A and CA. To date, a large number of MazF homologs that cleave RNA at specific three‐ to seven‐base sequences have been identified from bacteria to archaea. MazF‐ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate‐binding site. Here, we investigated the role of the two loops in MazF‐ec, which are closely associated with the interface of the MazF‐ec dimer. We examined whether exchanging the loop regions of MazF‐ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF‐mx) and MazF from Mycobacterium tuberculosis (MazF‐mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF‐ec with loop 2 regions from either MazF‐mx or MazF‐mt3 created a new cleavage sequence at (A/U)(A/U)AA and C in addition to the original cleavage site, A and CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA and C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Mutations in the Escherichia coli23S rRNA Increase the Rate of Peptidyl-tRNA Dissociation from the Ribosome

We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme

The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9 > dA10 > dC25 approximately dA10 plus dC25. The results are interpreted in terms of an interaction between the ribose ring of C25 and the ribose and base of A10, in agreement with the proposal of Ryder and Strobel [Ryder, S. P., and Strobel, S. A. (1999) J. Mol. Biol. 291, 295-311]. In general, there is a correlation between the ability to undergo ion-induced folding and the rate of ribozyme cleavage. An exception to this is provided by G8, for which substitution with uridine leads to severe impairment of cleavage but folding characteristics that are virtually unaltered from those of the natural species. This is consistent with a direct role for the nucleobase of G8 in the chemistry of cleavage.     

A role for U2/U6 helix Ib in 5' splice site selection.

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

Mutational analysis of splicing activities of ribonucleotide reductase α subunit protein from lytic bacteriophage P1201

A CP1201 RIR1 intein is found in the ribonucleotide reductase alpha subunit (RNR α subunit) protein of lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078. This intein can be over-expressed and spliced in Escherichia coli NovaBlue cells. Mutations of C539, the N-terminal residue of the C-extein in the CP1201 RIR1 protein, led to the changes of pattern and level of protein-splicing activities. A G392S variant was found to be a temperature-sensitive protein with complete splicing activity at 17 and 28°C but not at 37°C or higher. We also found that the cleavage at the CP1201 RIR1 intein C-terminus of the double mutant G392S/C539G was blocked, but other cleavage activities could be efficiently performed at 17°C. G392S/C539G variant possessed the properties of low-temperature-induced cleavage at the intein N-terminus.     

IL-10 promoter single nucleotide polymorphisms are significantly associated with resistance to leprosy

The minor haplotype −3575A/-2849G/-2763C in IL-10 promoter has been defined as a marker of disease resistance to leprosy and its severity in Brazilian population. Our investigation of six single-nucleotide polymorphisms (SNPs) in IL-10 promoter in 282 Indian leprosy patients and 266 healthy controls by direct PCR sequencing, however, showed that the extended haplotype: −3575T/-2849G/-2763C/-1082A/-819C/-592C was associated with resistance to leprosy per se and to the development of severe form of leprosy, using either a binomial (controls vs cases, P=0.01, OR=0.58, CI=0.37–0.89) or ordinal (controls vs paucibacillary vs multibacillary, P=0.004) model. Whereas, IL-10 haplotype −3575T/-2849G/-2763C/-1082A/-819T/-592A was associated with the risk of development of severe form of leprosy (P=0.0002) in contrast to the minor risk haplotype −3575T/-2849A/-2763C in the Brazilian population. The role of IL-10 promoter SNPs in Brazilian and Indian population strongly suggests the involvement of IL-10 locus in the outcome of leprosy.     

Crystal structure of guanylyl-2′,5′-cytidine dihydrate: An analogue of msDNA-RNA junction in stigmatella aurantiaca

Sequence analysis of msDNA from bacterium such as Stigmatella aurantiaca, Myxococcus xanthus and Escherichia coli B revealed that the guanine residue of the single-stranded RNA is linked to the cytosine residue of the msDNA through a 2′–5′ instead of a conventional 3′–5′ phosphodiester bond. We have now obtained the crystal structure of the self-complementary dimer guanylyl-2′,5′-cytidine (G2′p5′C) that occurs at the msDNA-RNA junction. G2′p5′C crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 8.376(2), b = 16.231(5), c = 18.671(4). CuK ∝ intensity data were collected on a diffractometer in the ω ?2θ scan mode. The amount of 1699 out of 2354 reflections having I ≥ 3σ (F) were considered observed. The structure was solved by direct methods and refined by full-matrix least squares to a R factor of 0.054. The conformation of the guanine base about the glycosyl bond is syn (χ₁ = ?54°) and that of cytosine is anti (χ₂ = 156°). The 5′ and 2′ and ribose moieties show C2′-endo and C3-endo mixed puckering just like in A2′p5′A, A2′p5′C, A2′p5U, and dC3′p5′G. Charge neutralization in G2′p5′C is accomplished through protonation of the cytosine base. An important feature of G2′p5′C is the stacking of guanine on ribose 04′ of cytosine similar to that seen in other 2′–5′ dimers. G2′p5′C, unlike its 3′–5′ isomer, does not form a miniature double helix with the Watson-Crick base-pairing pattern. Comparison of G2′p5′C with A2′p5′C reveals that they are isostructural. A branched trinucleotide model for the msDNA-RNA junction has been postulated. © 1994 John Wiley & Sons, Inc.     

Nucleotide sequence of Scenedesmus obliquus cytoplasmic initiator tRNA.

The cytoplasmic initiator tRNA from the green alga Scenedesmus obliquus has been purified and its sequence shown to be p A G C U G A G-U m G m G C G C A G D G G A A G C G psi m G A psi G G G C U C A U t A A--C C C A U A G m G D m C A C A G G A U C G m A A A C C U Gm U C U C A--G C U A C C A-O H. The sequence has been deduced and confirmed using several different P-post labelling techniques. The sequence is similar to those of other eukaryotic cytoplasmic initiator tRNAs and it has the sequence G A U C in place of the usual G T psi C. Although it resembles lower eukaryotic species in having a U preceding the anticodon and a modified G in the T psi C stem, in overall homology it is closer to the higher eukaryotic than to the fungal initiator tRNAs.     

Spectinomycin interacts specifically with the residues G1064 and C1192 in 16S rRNA, thereby potentially freezing this molecule into an inactive conformation.

The upper stem of helix 34, consisting of the base-paired sequences C1063G1064U1065 and A1191C1192G1193, is suggested to be involved in the binding of spectinomycin. In E. coli 16S rRNA, each of the three mutations at position C1192 confers resistance to spectinomycin. In chloroplast ribosomes from tobacco plants and algae, resistance is conferred by single mutations at positions 1064, 1191, and 1193 (E. coli numbering). Since each of these mutations disrupt any of the three basepairs in the upper stem of helix 34, it has been postulated that spectinomycin can bind to this region and inhibit protein synthesis, only if its nucleotides are basepaired. We have tested this hypothesis by introducing disruptive and compensatory mutations that alter the basepair G1064-C1192. Using the specialized ribosome system, the translational activity of such mutants was determined, in the absence and presence of spectinomycin. We show that any of the three disruptive mutations A1064, C1064, and U1064 confer resistance, in accordance with the model for spectinomycin binding. Compensatory mutations A1064U1192, C1064G1192, and U1064A1192, however, maintained the resistance. This indicates that a basepaired conformation as such is not sufficient for spectinomycin binding, but rather that a G-C pair at positions 1064 and 1192 is required. In addition, we find that the translational activity of specialized ribosomes containing the mutations C1064G1192 is 5-fold lower compared to that of ribosomes containing any of the other mutations introduced, regardless whether spectinomycin is present or not. Since the introduction of C1064G1192 is expected to increase the stability of the upper stem of helix 34, we suggest that these mutations impair ribosome function by preventing the (transient) disruption of the upper stem. By analogy, we speculate that spectinomycin blocks protein synthesis by stabilizing the upper stem. In both cases, the 30S subunit would be frozen into an inactive conformation.