Paul J. Romaniuk

Computer modeling from solution data of spinach chloroplast and of Xenopus laevis somatic and oocyte 5 S rRNAs

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms.

Joining of RNA molecules with RNA ligase.

Publisher Summary T4 RNA ligase catalyzes the ATP-dependent phosphodiester bond formation between a 5´-terminal phosphate (donor) and a 3´-terminal hydroxyl (acceptor). Although the enzyme can produce circular products with substrates of sufficient length that contain the required termini RNA ligase is also a very effective intermolecular joining reagent for oligonucleotide synthesis. The versatility of RNA ligase as a reagent for oligoribonucleotide synthesis is illustrated by the following two examples. For preparative synthesis, the enzyme readily catalyzes the equimolar joining of two oligoribonucleotide blocks on a moderate scale (ca. 1 μmol) and the enzyme can be used to synthesize small amounts of internally P-labeled oligomers of high specific activity. This chapter discusses applications for oligoribonucleotide synthesis and also explains the use of RNA ligase for the preparation of deoxy oligomers.

Interaction of R17 Coat Protein With Its RNA Binding Site For Translational Repression

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kd at 2 degrees C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of Ka and a delta H = -19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RNAs failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific. We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.

Rapid identification of Campylobacter species using oligonucleotide probes to 16S ribosomal RNA

A comparison of the 16S ribosomal RNA sequences from various Campylobacter species was used to identify unique sequences which distinguish pathogenically significant campylobacters from other members of the genus. Oligonucleotides complementary to these sequences were synthesized, and under stringent conditions hybridization reactions using these probes and total RNA as the target nucleic acid displayed the desired species specificity. A simple, rapid lysis procedure was developed that allowed the specific detection of C. jejuni or C. coli from as few as 10(6) bacterial cells in less than eight hours. This method provides immediate advantages for the unequivocal identification of Campylobacter species in the microbiology laboratory, and demonstrates the potential for the use of these probes in a rapid diagnostic test of clinical samples for Campylobacter infection.

Involvement of “hinge” nucleotides of Xenopus laevis 5 S rRNA in the RNA structural organization and in the binding of transcription factor TFIIIA☆

Nucleotides in the bifurcation region of the 5 S rRNA, the junction of the three helical domains, play a central role in determining the coaxial stacking interactions and tertiary structure of the RNA. We have used site-directed mutagenesis of Xenopus laevis oocyte 5 S rRNA to make all possible nucleotide substitutions at three positions in loop A (10, 11 and 13) and at the G66.U109 base-pair at the beginning of helix V. Certain double point mutations were constructed to ascertain the relationship between loop A nucleotides and the G.U base-pair. The importance of the size of the bifurcation region was tested by the creation of a single nucleotide deletion mutant and two single nucleotide insertion mutants. The effects of these mutations on the structure and function of the 5 S rRNA were determined by solution structure probing of approximately half of the mutants with chemical reagents, and by measuring the relative binding affinity of each mutant for transcription factor TFIIIA. Proposed structural rearrangements in the bifurcation region were tested by using a graphic modeling method combining stereochemical constraints and chemical reactivity data. From this work, several insights were obtained into the general problem of helix stacking and RNA folding at complex bifurcation regions. None of the mutations caused an alteration of the coaxial stacking of helix V on helix II proposed for the wild-type 5 S rRNA. However, the formation of a Watson-Crick pair between nucleotide 13 of loop A and nucleotide 66 at the top of helix V does cause a destabilization of the proximal part of this helix. Also, nucleotide 109 at the top of helix V will preferentially pair with nucleotide 10 of loop A rather than nucleotide 66 when both possibilities are provided, without affecting the stability of helix V, even though the G.U pair is disrupted. The effects of these mutations on TFIIIA binding indicate that the bifurcation region is critical for protein recognition. One important feature of the relationship between 5 S rRNA structure and TFIIIA recognition resulting from this study was the observation that any mutation that constrains the bifurcation loop results in a reduced affinity of the RNA for TFIIIA, unless it is compensated for by an increased flexibility elsewhere.

Ribosomal 5S RNA from Xenopus laevis oocytes: conformation and interaction with transcription factor IIIA.

This review describes extensive studies on 5S rRNA from X laevis oocytes combining conformational analyses in solution (using a variety of chemical and enzymatic probes), computer modeling, site-directed mutagenesis, crosslinking and TFIIIA binding. The proposed 3-dimensional model adopts a Y-shaped structure with no tertiary interactions between the different domains of the RNA. The conserved nucleotides are not crucial for the tertiary folding but they maintain an intrinsic structure in the loop regions. The model was tested by the analysis of several 5S rRNA mutants. A series of 5S RNA mutants with defined block sequence changes in regions corresponding to each of the loop regions was constructed by in vitro transcription of the mutated genes. Our results show that none of the mutations perturbs the Y-shaped structure of the RNA, although they induce conformational changes restricted to the mutated regions. The interaction of the resulting 5S rRNA mutants with TFIIIA was determined by a direct binding assay. Only the mutations in the hinge region between the 3 helical domains have a significant effect on the binding for the protein. Finally, TFIIIA was crosslinked by the use of trans-diamminedichloroplatinum (II) to a region covering the fork region. Our results show that (i) the tertiary structure does not involve long-range interactions; (ii) the intrinsic structures in loops are strictly sequence-dependent; (iii) the hinge nucleotides govern the relative orientation of the 3 helical domains; (iv) TFIIIA recognizes essentially specific features of the tertiary structure of 5S rRNA.

Effect of mutations in domain 2 on the structural organization of oocyte 5 S rRNA from Xenopus laevis

In order to test and refine the molecular model of Xenopus laevis 5 S rRNA proposed in a previous work, we have synthesized, by site-directed mutagenesis and in vitro transcription, four mutants in the internal loop B and in the hairpin loop C of domain 2. The conformations of these mutant 5 S rRNAs have been tested using a variety of enzymatic and chemical structure-specific probes and computer modeling. The mutations induce conformational changes restricted to the mutated regions. Our results demonstrate unambiguously that the three helical domains of the Y-shaped structure are independent and that loop C possesses an intrinsic conformation, which is not involved in any tertiary long-range interaction. They point to the crucial role of invariant nucleotides in maintaining the intrinsic conformation of the loop and to the effect of sequence on the stability of loop regions.

Retinoid X Receptor Alters the Determination of DNA Binding Specificity by the P-box Amino Acids of the Thyroid Hormone Receptor

Nuclear hormone receptors bind to hormone response elements in DNA consisting of two half-sites of 6 base pairs. The P-box amino acids of each receptor determine the identities of the central nucleotides of the half-site. 57 P-box variants of the human thyroid hormone receptor (hT3Rβ) were used to demonstrate the relationship between P-box sequence and DNA binding specificity by homodimers and heterodimers formed with the retinoid X receptor (RXR). In general, the formation of heterodimers relieved many of the constraints on the compatibility of hT3Rβ P-box sequences with DNA binding. Effects were most dramatic for heterodimers bound to a direct repeat spaced by four base pairs. RXR also overrides the P-box-derived DNA binding specificity of hT3Rβ when heterodimers are bound to inverted or everted repeat elements. These effects of RXR are most pronounced on AGGCA half-sites but are squelched when the RXR partner of the heterodimer is bound to an AGGCA half-site. The influence of RXR on hT3Rβ DNA binding specificity varies with the orientation of half-sites in the element, the identity of the fourth base pair of the half-site, and the spacing between the half-sites of direct repeats. These differences suggest that the DNA binding domains of RXR-hT3Rβ heterodimers are not positioned equivalently on the various elements, affecting the manner in which the P-box amino acids of hT3Rβ interact with base pairs within the half-site.

Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.