Alternative Secondary Structures Research Articles

Recent insights on RNA folding mechanisms from catalytic RNA.

Methods for probing RNA structure in real time have revealed that initial folding steps are complete in less than a second. Refolding of large catalytic RNAs in vitro often results in long-lived intermediates that reach the native structure very slowly. These kinetically trapped intermediates arise from alternative secondary structures that form early in the folding process. In cells, proteins modulate the outcome of RNA folding reactions by stabilizing specific conformations or by accelerating refolding of misfolded intermediates. At the same time, competition between metastable conformations provides a means for regulating the biological activity of transcripts.

Nef/long terminal repeat quasispecies from HIV type 1-infected Mexican patients with different progression patterns and their pathogenesis in hu-PBL-SCID mice.

To examine the genetic features of the long terminal repeat (LTR) derived from six HIV-1-infected individuals enrolled in the Mexico City Cohort, we cloned and sequenced a 505-bp fragment of the proviral LTR from their peripheral blood mononuclear cells (PBMCs). All patients harbored HIV-1 LTR quasispecies corresponding to the B subtype. Three patients with high CD4+ T cell counts (>500/mm3) presented LTR sequences with point mutations in the TAR bulge. The LTR sequence from a patient classified as a long-term nonprogressor (LTNP) presented the most frequent naturally occurring length polymorphism (MFNLP) and two substitutions in the TAR region that were predicted to result in two alternative secondary RNA structures. A novel 18-bp deletion, which eliminates part of the putative binding site for the nuclear factor of activated T cells (NFAT-1), was identified in the overlapping nef/LTR sequence derived from a patient progressing to AIDS. This deletion coincides with the ability of this virus to consistently replicate at low levels in vivo (viral load <500 RNA copies/ml) and in vitro (unsuccessful virus isolation). On one occasion, when virus isolation was successful, the 18-bp deletion was no longer evident and LTR sequences with intact NFAT-1-binding sites were observed. Inoculation of hu-PBL-SCID mice with viruses from several Mexican patients resulted in differential CD4+ T cell depletion patterns 15 days postinfection, which agree with the in vivo CD4+ T cell count data from each patient.

In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase.

In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis.

Evolutionary, Mechanistic, and Predictive Analyses of the Hydroxymethyldihydropterin Pyrophosphokinase Family of Proteins

A prediction has been preparedab initiofor the secondary structure of the hydroxymethyldihydropterin pyrophosphokinase (HPPK) family of proteins starting from a set of aligned homologous protein sequences. Attempts to identify a fold by threading failed, judging by the inability to find a threading “hit” that had a secondary structure that was plausibly congruent to the predicted secondary structure for the HPPK family. Therefore, a set of tertiary structure models was assembledab initio,where alternative models were built and used to select between alternative secondary structure models. This prediction report illustrates the importance of non-computational approaches to structure prediction at its present frontier, which is to obtain medium resolution models of tertiary structure.

Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme.

The free energy of substrate binding to the hammerhead ribozyme was compared for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected because neither ribozyme nor substrate oligonucleotide formed detectable alternate secondary structures. The observed free energies of binding varied from -8 to -24 kcal/mol and agreed very well with binding energies calculated from the nearest-neighbor free energies if a constant energetic penalty of DeltaG degreescore = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure showed weaker binding to the ribozyme by an amount consistent with the predicted free energy for hairpin formation. These thermodynamic conclusions permit the prediction of substrate binding affinities for ribozyme-substrate pairs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.

Ironing out the kinks: splicing and translation in bacteria.

In eukaryotic cells, RNA processing is physically separate from protein synthesis. As nuclear export of unspliced RNA is restricted, only mature messages are normally exposed to the translational machinery. In bacteria, however, splicing must be coordinated with the translation of nascent transcripts. These two processes make very different demands on the RNA substrate: Splicing of autocatalytic introns requires that the 58 and 38 splice sites be brought together as part of an elaborate tertiary structure, whereas translation requires that the mRNA be relatively free of secondary structure. Nonetheless, introns have been found in highly expressed genes in eubacteria, bacteriophages, mitochondria, and chloroplasts (for review, see Burke 1988). Clearly, there must be some means of balancing splicing of bacterial introns with cotranscriptional translation. In this issue, Semrad and Schroeder (1998) provide the surprising answer that splicing of a group I intron from phage T4 is facilitated by translation of the upstream open reading frame (ORF). This enhancement of splicing is achieved by modulating the long-range conformation of the premRNA. Their results provide useful analogies for the coupling of eukaryotic pre-mRNA splicing with transcription. Bacterial mRNAs exclusively contain group I or group II introns, and the three group I introns that are present in phage T4 are all able to self-splice in vitro (for review, see Belfort 1990). The introns are found in genes encoding thymidylate synthase (td), ribonucleotide reductase (nrdB) (Belfort 1990), and anaerobic ribonucleotide reductase (sunY, or nrdD) (Young et al. 1994). In addition to sequences that provide the necessary functions of selfsplicing, td and sunY in trans contain internal ORFs that encode double-stranded DNA endonucleases (Belfort 1989). The endonucleases trigger homing, or site-specific movement of the intron sequences to intronless alleles. Self-splicing requires that the intron RNA fold into a unique secondary and tertiary structure (Cech and Herschlag 1996). The central core of this structure is highly conserved among group I introns and contains the active site where the transfer of phosphodiester bonds takes place. A helix containing the 58 splice site docks into the active site via hydrogen bonds with its ribose 28 hydroxyl groups (Pyle et al. 1992). Recognition of the 38 splice involves several weak interactions, including a 2-bp stem between the 38 end of the intron and nucleotides in the intron core (Burke et al. 1990). The folded structure of the RNA depends on coordination of magnesium ions, which are required for self-splicing (Cech and Herschlag 1996). Long-range interactions, such as base-pairing between hairpin loops, or tetraloop–helix receptor interactions, also stabilize the tertiary structure of the catalytic core (for review, see Brion and Westhof 1997). Recent experiments on the folding kinetics of group I introns, as well as experiments carried out in the 1970s on tRNA, have begun to tease out the mechanisms by which RNAs reach a biologically active conformation (for review, see Draper 1996; Brion and Westhof 1997). Small hairpins can form in 10–100 μsec, and tRNAs can fold within milliseconds (Draper 1996). In contrast, larger RNAs fold in stages over much longer periods of time. The tertiary structure of the P4–P6 domain of the Tetrahymena group I intron, which can fold independently, appears in a few seconds (Sclavi et al. 1997). The core of the intron, however, takes several minutes to become completely folded (Zarrinkar and Williamson 1994; Banerjee and Turner 1995). The very slow folding of longer RNAs arises, in part, from their tendency to form many alternative secondary structures. As RNA secondary structure is stable, incorrect base pairs have the potential to trap the molecule in inactive conformations that can persist for relatively long periods of time (for review, see Herschlag 1995; Thirumalai and Woodson 1996; Brion and Westhof 1997). These metastable states may be quite dissimilar to the final structure. Therefore, the folding process itself can result in RNA populations with different levels of biological activity. Misfolding of RNA has been shown to inhibit ribozyme activity and spliceosome assembly (Goguel et al. 1993; Zavanelli et al. 1994; Uhlenbeck 1995). As discussed below, competition between metastable RNA conformations can also serve as a normal mechanism by which gene activity is regulated. Estimates of in vivo splicing rates are 10to 50-fold faster than in vitro self-splicing (Brehm and Cech 1983; Zhang et al. 1995). This disjunction between in vitro and in vivo activity of catalytic RNAs implies that kinetic folding traps are normally overcome in the cell. Proteins E-MAIL sw74@umail.umd.edu; FAX (301) 314-9121.

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

CTG repeats associated with human genetic disease are inherently flexible

The lengthening of tracts of CTG, CGG and GAA triplet repeats during progression of a pedigree has been associated with more than 12 human genetic diseases, including fragile X syndrome, myotonic dystrophy and Friedreich’s ataxia. These repetitive sequence elements have the potential to form alternative DNA secondary structures that may contribute to their instability. The alternative DNA secondary structures may mediate errors during DNA replication, repair or recombination of the triplet repeat, leading to expansion. Here we show that DNA composed of pure CTG or CGG repeats exhibits anomalously fast mobility on polyacrylamide gels, confirming a previous observation for DNA containing CTG and CGG triplet repeats flanked by mixed sequence DNA. Moreover, we show that even short tracts of duplex CTG repeats have an unusual helix structure. CTG repeats reduce overall curvature associated with phased A-tract or GGCC curves, but alone they do not introduce curvature into DNA. The reduction in curvature of phased A-tracts by CTG repeats is similar to that afforded by an interspersed flexible region associated with a (TT)·(TT) mispair. CTG-containing DNAs exhibit a rapid rate of cyclization, consistent with a flexible helix. These results suggest that tracts of (CTG)·(CAG) repeats are inherently flexible. In addition, our results suggest that the unusual rapid electrophoretic mobility of CTG- or CGG-containing DNA may be a consequence of an extended flexible DNA chain.

DNA (cytosine-5)-methyltransferases in mouse cells and tissues. studies with a mechanism-based probe

The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-specific DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identified. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identifiable de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not fit the definition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence specificity of intact Dnmt1 in whole-cell lysates. Specificity was found to be confined to the sequence 5′-CpG-3′; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-specific de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.

Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.

Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.

A revised secondary structure model for the 3'-end of hepatitis B virus pregenomic RNA.

The polymerase encoded by human hepatitis B virus, which has reverse transcriptase and RNase H activity, binds to its pregenomic RNA template in a two-step process involving a terminal redundancy. Both first strand and second strand DNA synthesis involve primer translocation and second strand synthesis involves a template jump. Three parts of the genome, including the so-called core promoter, are known to show deletions in strains usually arising after long-standing HBV infection, but also in some patients treated with interferon. A computer-based study of RNA template folding in the core promoter region, accommodating well-known point mutations, has generated a model for the 3' DR1 primer binding site as being part of a superstructure encompassing an already well-established stem-loop. Depending on the identity of nucleotides 1762 and 1764, the DR1 region may assume two alternative secondary structures which stabilize it as a primer binding site to different extents. Remarkably, one of these structures includes a pronounced loop which coincides with at least 12 related deletions seen in HBV DNA from different patients. Thus according to the model, the 5'- and 3'-ends of pregenomic RNA, which share primary sequences but have separate functions, are not structural equivalents. An RNA superstructure near the 3'-end of all HBV transcripts could have far-reaching implications for the modulation of both genome replication and post-transcriptional processing.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci.

Most models proposed to explain the disease-associated expansion of (CTG)n.(CAG)n and (CGG)n.(CCG)n trinucleotide repeats include the formation of slipped strand DNA structures during replication; however, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystrophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premutation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing equal lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures are characterized by reduced electrophoretic mobilities in polyacrylamide gels. These alternative secondary structures are stable at physiological ionic strengths and to temperatures up to at least 55 degrees C. Following reduplexing, the CAG strand of the (CTG)n.(CAG)n repeats is preferentially sensitive to mung bean nuclease, suggesting the presence of single-stranded DNA in the alternative DNA structure. For (CTG)17, which is a repeat length found in normal individuals, less than 3% of the DNA molecules formed alternative DNA structures upon reduplexing. DNA molecules containing (CTG)50 or (CTG)255, which represent premutation and full mutation lengths of triplet repeats, respectively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed increased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the novel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential involvement of these structures, which we term S-DNA, in the gentic instability of triplet repeats is discussed.

Redox-Triggered Secondary Structure Changes in the Aggregated States of a Designed Methionine-Rich Peptide

We have previously shown that methionine can be used as a “switchable” residue for the design of peptides with alternative secondary structure preferences in the aggregated state (J. Am. Chem. Soc. 1993, 115, 12609). Redox-induced secondary structure changes in the 18-residue peptide Ac-YLKAMLEAMAKLMAKLMA-NH2 result from conversion of lipophilic methionine (M) to hydrophilic methionine sulfoxide (M°), which transforms a peptide capable of adopting an amphiphilic α-helical conformation into a peptide capable of adopting an amphiphilic β-strand conformation. Here we present a detailed characterization of the third oxidation state of this peptide, in which the methionine residues are oxidized to the sulfone state. The sulfone form behaves similarly to the sulfoxide form, even though the sulfone group is somewhat less hydrophilic than the sulfoxide group. These results provide support for the concept that the conformational preferences of peptides and proteins are strongly dependent upon the linear ordering of hydrophilic and lipophilic residues (“amphiphilic order”).

RNA Chaperones and the RNA Folding Problem

RNA Chaperones and the RNA Folding Problem

Structural Changes in Base-paired Region 28 in 16 S rRNA Close to the Decoding Region of the 30 S Ribosomal Subunit are Correlated to Changes in tRNA Binding

Escherichia coli30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921–922)·(1395–1396) and (923–925)·(1391–1393), which are part of region 28, are unstable and an alternate arrangement, (921–923)·(1532–1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC-SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity.

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA

A convenient small scale synthesis has been developed for obtaining peptide nucleic acid oligomers (PNAs). PNAs have been conjugated to a protein, staphylococcal nuclease, through disulfide exchange between a cysteine at the 3′-(carboxy) end of the PNA and an introduced cysteine on the surface of the nuclease. Site specific DNA cleavage by the attached nuclease has been used to examine the Watson-Crick hybridization of the PNAs to duplex DNA. Substantial affinity cleavage occurred when target sites contained inverted repeats which have the potential to form non B-DNA structures such as cruciforms. No affinity cleavage was observed at a site lacking apparent potential for non B-DNA structures. These results indicate that the Watson-Crick hybridization of PNAs to duplex DNA by strand displacement is favored by the presence of potential alternative secondary structures within the target sequence.

Regulation of int gene expression in bacteriophage P2

The int gene of bacteriophage P2 is the only viral gene necessary for the integration of P2 into the Escherichia coli host chromosome. This gene is situated between the phage attachment site, attP, and the repressor C gene, and is cotranscribed with C from the Pc promoter, towards attP. The Pc promoter is negatively controlled by the cox gene, which is the first gene of the early operon. In vitro recombination assays have indicated that in P2 an overproduction of Int is deleterious to the integrative process. We report here that the level of int expression is affected by several different mechanisms after transcriptional initiation. First, a partial transcription termination signal located between the int and C genes reduces the the transcriptional readthrough by about 30%. Second, the ribosome binding site and AUG codon of the int gene are located in a putative stem-loop structure, which may inhibit the initiation of translation. The nip1 mutation (a G to A substitution at the 22nd coding nucleotide of int which results in an increased efficiency of excision) is shown to relieve this inhibition, possible through the formation of an alternative mRNA secondary structure. However, the third and probably most important control of int expression in P2 seems to be that of posttranscriptional autoregulation. The binding site of the Int protein on int gene mRNA is shown to extend into the ribosome binding site of int, supporting our earlier proposed model of competitive binding between Int and ribosomes.