Terminal Guanosine Residue Research Articles

Enhancing DNA Crystal Durability through Chemical Crosslinking.

Three-dimensional (3D) DNA crystals have been envisioned as a powerful tool for the positional control of biological and non-biological arrays on the nanoscale. However, most DNA crystals contain short duplex regions that can result in low thermal stability. Additionally, because DNA is a polyanion, DNA crystals often require high cation concentrations to maintain their integrity. Here, we demonstrate that a DNA alkylating mustard, bis(2-chloroethyl)amine, can form interstrand crosslinks within a model 3D DNA crystal. The crosslinking procedure did not alter crystal X-ray diffraction properties, but it did significantly improve the overall stability of the crystals under a variety of conditions. Crosslinked crystals showed enhanced stability at elevated temperature and were stable at Mg(2+) concentrations as low as 1 mm. Remarkably, the crosslinked crystals showed significant resistance to DNase I treatment, while also having improved longevity in tissue culture mediums. Characterization of the crosslinked species suggest that there are multiple crosslinking sites, but that the most prevalent interstrand crosslink involves an unpaired 3'-terminal guanosine residue. The improved stability of these DNA crystals suggests that simple treatment with alkylating reagents might be sufficient to stabilize crystals and other DNA constructs for improved functionality in biological and non-biological applications.

Labeled Primers for Mutation Scanning: Making Diagnostic Use of the Nucleobase Quenching Effect

The advent of the PCR has revolutionized many molecular biological techniques, including single-nucleotide polymorphism (SNP) detection. Interest in the detection and discovery of human polymorphisms increased further with the complete sequencing of the human genome. The main applications for SNP detection include diagnosis of genetic disease and identification of functional mutations in genes that are of pharmacogenetic importance. The results from testing for somatic mutations remain with a person for a lifetime, and retesting is performed only rarely. To be able to deliver a molecular diagnosis of the highest quality, we must have a sound understanding of the different principles used for the detection of polymorphic DNA. The clinical laboratory today is equipped with a plethora of assays for the detection of SNPs, and new detection methods continue to be published. Molecular diagnostic assays often make use of the real-time monitoring of hybridization for the detection of base mutations. A common assay set-up uses a fluorescence resonance energy transfer (FRET) probe combination. A 3′-dye-labeled detection probe together with a 5′-dye-labeled and 3′-phosphorylated anchor probe interact by FRET in the hybridized state. In the unhybridized state, no FRET occurs, making this technique applicable to homogeneous single-tube assays. Variations of this theme include the use of exonuclease probes and molecular beacons. Initially it was recommended to avoid attaching the dye to a terminal guanosine residue because quenching was observed (1), but for molecular beacons, a design modification was suggested. Instead of the second dye or quencher, a string of guanosine bases was opposed …

An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron.

On the basis of the nucleotide sequence of Tetrahymena group I intron, we constructed a 31 residue RNA that has the P7 stem and the 3'-terminal guanosine residue (3'-G) with a putative stem-loop structure (P9.0) intervening between them. For this model RNA (P7/P9.0/G), four residues around the guanosine binding site (GBS) in the P7 stem were found to exhibit much lower sensitivities to ribonuclease V1 than those of a variant RNA having adenosine in place of the 3'-G, suggesting that the 3'-G contacts around the GBS. NMR analyses of the imino proton resonances of the P7/P9.0/G RNA indicated that the base pairing in the GBS is retained on the interaction with the 3'-G, and that the two base pairs of the putative P9.0 stem-loop are definitely formed. Comparison of the RNA with its variants with either A (3'-A) or a deletion in place of the 3'-G suggested that the stability of the P9.0 stem-loop is affected by the GBS-3'-G interaction. The melting temperatures of the P9.0 stem-loop were determined from the UV absorbances of these RNAs, which quantitatively indicated that the P9.0 stem-loop is significantly stabilized by the interaction of the GBS with the 3'-G, rather than the 3'-A, and also by direct interaction with divalent cations (Mg2+, Ca2+ or Mn2+). Upon replacement of the G-C base pair by C-G in the GBS of the P7/P9.0/G RNA, the specificity was switched from 3'-G to 3'-A, as in the case of the intact intron.

Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3′ splice site

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3′-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3′ splice site. This circle is formed at the phosphodiester bond of the 5′-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3′-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3′ splice site, restoring a guanosine to the 3′ terminus and re-establishing P9.0 pairing. The new 3′ terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3′-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.

Possible involvement of (2'5')oligoadenylate synthetase activity in pre-mRNA splicing.

The first step in splicing of pre-mRNA involves an intermediate lariat structure, in which a 2'5' phosphodiester bond between the 5' terminal guanosine residue of the intron and a specific adenosine residue near the 3' end of the intron is formed. A mammalian enzyme that generates 2'-5' phosphodiester bonds is (2'-5')oligoadenylate synthetase [(2'-5')OASE]. Although the expression of this enzyme is induced by interferon, low constitutive levels can be detected in untreated cells and tissues. The structural similarity between the lariat branch point and the 2'-5' phosphodiester bond generated by (2'-5')OASE prompted the experiments described here which suggest that this enzyme is involved in pre-mRNA splicing. (i) We show that a (2'-5')OASE activity is associated with 60S spliceosomes in an ATP- and RNA-dependent manner and that it can be indirectly immunoprecipitated by anti-Sm antibodies. (ii) Antibodies against (2'-5')OASE inhibit the lariat formation in the first step of splicing when added directly to a splicing reaction in vitro. (iii) HeLa cell nuclear extracts immunodepleted of (2'-5')OASE activity were also deficient in splicing activity.

New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing

The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5′ splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5′ and 3′ splice sites independently show that splicing occurs in two separable steps. The 3′ splice site was found to be susceptible to site-specific hydrolysis leaving a 3′ hydroxyl terminus. This is interpreted as an indication that the 3′ splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5′ splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: (1) transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); (2) transesterification by oligopyrimidines or by the 5′ exon (which terminates with C-U-C-U-C-U OH) at the site following the 3′-terminal guanosine residue of the intervening sequence; and (3) specific hydrolysis at the splice sites. One of the products of the reactions at the 3′ splice site is a molecule that contains the 5′ exon still attached to the intervening sequence. It has a 3′-terminal G OH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5′ splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3′-terminal G of the intervening sequence.

RNA splicing in Neurospora mitochondria. The large rRNA intron contains a noncoded, 5'-terminal guanosine residue.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.

Complementary addressed modification of oligonucleotide d(pGpGpCpGpGpA) with platinum derivative of oligonucleotide d(pTpCpCpGpCpCpTpTpT)

New heterobifunctional reagent [BrPt(dien)—(CH 2) 6—(dien)Pt(H 2O)] 3+ (I) (− dien = diethylenetriamine residue) is proposed for preparation of complementary addressed reagents, reactive derivatives of oligonucleotides and polynucleotides. Two reactive groupings of (I) bind to oligonucleotides at different rates due to the higher reactivity of −[(dien)Pt(H 2O)] 2+ as compared to −[(dien)Pt Br] +. Using (I), bromodiethylenetriaminoplatinum group was attached to the oligonucleotide d(pTpCpCpGpCpCpTpTpT) (II) by rapid reaction of aquatriaminoplatinum group of (I) with guanosine residue of (II). The reactive oligonucleotide derivative III thus obtained was shown to modify predominantly 5′-terminal guanosine residue in the complementary oligonucleotide d(pGpGpCpGpGpA) (IV).

Identification of a unique guanine-7-methyltransferase in Semliki forest virus (SFV) infected cell extracts

The methylation of the 5′ terminal guanosine residue of the cap structure of Semliki Forest virus (SFV) mRNAs has been shown to occur in vitro concomitantly with their synthesis ( R. K. Cross and P. J. Gomatos, Virology, 114, 542–554, 1981) . The enzyme responsible for this methylation, a guanine-7-methyltransferase, is associated with the SFV replication complex which contains both the virus-specified polymerase and RNA template in a mitochondrial pellet fraction, P-15, from infected cell lysates. In the present report, evidence has been obtained demonstrating that a virus-specified function is required for this methylating activity. First, the methyltransferase enzyme in these infected P-15 extracts has been found to differ in substrate specificity from that of the BHK host cell enzyme. This enzyme was able to catalyze the methylation of GTP to m 7GTP in vitro whereas the cellular enzyme could not methylate GTP. The incorporation of a methyl group onto GTP occurred linearly for at least 2 hr at 30° under conditions of neutral pH and added GTP substrate. Second, a study of the kinetics of appearance of this activity, has demonstrated that the capacity to methylate GTP did not appear until 1 hr after infection and reached maximal levels by about 3 hr. Third, de novo protein synthesis was required. Addition of the protein synthesis inhibitor, cycloheximide, prevented the appearance and subsequent increase in the methylating activity. However, once formed the methyltransferase was found to be stable for at least 3 hr. These results suggest that an early viral function, perhaps a nonstructural polypeptide is required for this novel guanine-7-methyltransferase activity in SFV infected cell extracts.

The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA.

The ribosomal RNA precursor of Tetrahymena thermophila contains a 0.4 kilobase intervening sequence that is excised as a linear RNA molecule ("IVS RNA") and subsequently cyclized. In vitro transcription in isolated nuclei was used to accumulate the IVS RNA labeled at its 5' end was subjected to sequencing gel analysis and terminal nucleotide analysis. In addition, uniformly labeled IVS RNA was cleaved with RNAase T1, and the resulting oligonucleotides were studied by two-dimensional fingerprinting. The IVS RNA was found to be a unique molecule with no discernible terminal heterogeneity. The 5'-terminal nucleotide is a guanosine that is not present at the corresponding point in the DNA sequence, determined by N. Kan and J. Gall (see adjoining paper). This nucleotide is added to the IVS during splicing [Cech, Zaug, and Grabowski (1981) Cell 27, 487-496]. Based on the sequences near the ends of the RNA, the remainder of the RNA sequence is colinear with that of the DNA. The IVS RNA has 5'-monophosphate and 3'-hydroxyl termini. Comparison of these results to those obtained previously for yeast tRNA intervening sequences leads us to conclude that the splicing mechanisms are fundamentally different for these two classes of transcripts.

Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells.

The 5' termini of many viral and cellular mRNAs contain sequences of the type m7G(5")pppNm. An RNA (guanine-7-)-methyltransferase that specifically methylates the 5'-terminal guanosine residue of RNAs ending in the dinucleoside triphosphate G(5')pppN- has been purified from the cytoplasm of HeLa cells. Approximately two-thirds of the methyltransferase activity detected in an assay employing umnethylated vaccinia virus mRNA as acceptor was located in the cytoplasm when cells were disrupted by Dounce homogenization; 30% of the cytoplasmic activity was associated with ribosomes but was removed by washing with 0.5 M KCl. The enzyme was purified 165-fold from the cytoplasm by removing nucleic acid by phase partition followed by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, denatured DNA-agarose, and CM-Sephadex. The partially purified enzyme preparation methylated heterologous tRNAs as well as vaccinia mRNA, but the tRNA methyltransferases could be separated from the mRNA activity by sucrose gradient sedimentation and gel filtration on Sephadex G-200. The product of the partially purified enzyme using vaccinia mRNA as substrate was exclusively 7-methylguanosine located in the terminal dinucleoside triphosphate. In addition to RNAs and synthetic polyribonucleotides terminating in a dinucleoside triphosphate, free G(5')pppG could be methylated but GTP, GDP, and G(5')pppG could not. The enzyme also methylated the dinucleoside diphosphate G(5')pppG but much less efficiently than G(5')pppG. An S20, W of 3.8, a Stokes radius of 3.6 nm, and a molecular weight of 56,000 were obtained from sucrose gradient sedimentation and Sephadex G-200 column chromatography.

M7G5'ppp5'GmptcpUp at the 5' terminus of reovirus messenger RNA.

In the presence of S-adenosyl methionine the 5' terminal guanosine residue of in vitro synthesized reovirus mRNA becomes methylated at the 2'-OH position. In addition, 7-methyl guanylic acid is condensed covalently at the 5' terminus resulting in the formation of a 5' to 5' triphosphate bridge. Analysis of the 5' terminal sequence of methylated reovirus mRNA revealed that it has the structure m7G5'ppp5'GmpCpUp.