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I Tinoco

Publications and source records attributed to I Tinoco.

At least 55 records · Page 3Linked to original sources
PubMed1996

Conformation of an RNA molecule that models the P4/P6 junction for group I introns.

We present a three-dimensional structure of a 34-nucleotide RNA molecule determined by NMR spectroscopy. The molecule was designed to form a junction between two double-helical stems whose sequence was based on the P4/P6 domain from group I introns. There are 5' and 3' single-strand overhangs at the junctions of the stems. Contrary to our expectations, we found that the 3' end of the molecule is placed in the minor and not the major groove of the P4 helix. As a result of tertiary contacts and stacking interactions from nucleotides in the 3' end, the junction helices are rotated in a left-handed fashion and do not stack coaxially. This conformation is highly dependent on the presence of single-stranded nucleotides at the 3' overhang. When the 3' end is removed, the molecule assumes a radically different structure with 5' end in the minor groove of the P6 helix and overall right-handed rotation between the stems. Only one nucleotide at the 3' end is sufficient to change the geometry of the junction.

Base Sequence↗
PubMed1995

Synthesis and NMR of RNA with selective isotopic enrichment in the bases.

Efficient syntheses of pyrimidine and purine nucleosides and nucleotides with selective 13C enrichment in the base moieties are described. Uridine and cytidine are labeled at position C6 and adenosine and guanosine are labeled at position C8. The selectively labeled nucleosides were converted to nucleoside triphosphates and used with in vitro transcription to synthesize labeled RNA. Isotope-edited 12C and 13C sub-spectra of a omega 1-1/2-X-filtered NOESY experiment are demonstrated to be useful for making resonance assignments and for deriving structural information in large (> 20 nt) RNA molecules. The labeled RNAs also allow heteronuclear J-couplings and relaxation parameters to be measured without complications from 13C-13C J-couplings.

Adenine↗
PubMed1995

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.

Base Sequence↗
PubMed1995

The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus.

The structure of a 34-nucleotide RNA pseudoknot that causes efficient -1 frameshifting in the messenger RNA of mouse mammary tumor virus has been investigated by NMR. Spectral assignment of the pseudoknot was facilitated by comparative NMR studies on the pseudoknot and on two smaller hairpin RNAs, and by using selective 13C labeling and 13C-edited NMR techniques. The three-dimensional structure of the pseudoknot has been determined. The frameshifter pseudoknot possesses structural features not observed in previously reported model pseudoknots. It has a compact structure with a pronounced bend at the junction of its G.C-rich stems. A single adenylate residue is intercalated between the two stems so that direct coaxial staking of the stems is not possible. The lack of an opposing nucleotide for the stacked, intervening adenylate creates a hinge in the pseudoknot. Most of the loop nucleotides are restrained by base staking interactions which keep the loops from adopting extended conformations. The sterically constrained loops direct the bending of the pseudoknot at the stem-stem junction. The roles of the intercalated adenylate and loop lengths in causing bending can explain their requirement for efficient frameshifting. Our NMR data also indicate that there are internal dynamics associated with the pseudoknot. The unique, compact structure and conformational flexibility of the pseudoknot may be required for recognition and favourable interaction with the translating ribosome, or with translation factors associated with the ribosome.

Base Sequence↗
PubMed1995

Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.

Animals↗
PubMed1995

RNA structure at high resolution.

Studies of RNA structural motifs at high resolution by NMR and X-ray crystallographic methods have provided many insights into the fundamental forces that give rise to the unique structural characteristics of RNA. Non-Watson-Crick purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine base pairing, as well as base-phosphate and base-ribose hydrogen bonding, are important forces for folding and stabilizing RNA structures. Base stacking is as important in determining RNA conformations as hydrogen bonding interactions. With the noncanonical interactions, many single-stranded loop regions such as hairpin loops, bulge loops, and internal loops fold into well-defined secondary structures. Loop-loop and loop-helix interactions can produce tertiary structures such as pseudoknots. Also, single strands adjacent to helical regions can form tertiary contacts with base-paired nucleotides of the helices. As we learn more about the structures of the important motifs we can ask more specific questions about the mechanisms of RNA-mediated functions. Conformational flexibility rather than a specific shape of the RNA may be important for some biological reactions. However, knowledge of the structures and the ease of conformational change of the molecules involved in any process are essential for understanding and eventually controlling the process.

Base Sequence↗
PubMed1994

Characterization of a "kissing" hairpin complex derived from the human immunodeficiency virus genome.

Base-pair formation between two hairpin loops--a "kissing" complex--is an RNA-folding motif that links two elements of RNA secondary structure. It is also a unique protein recognition site involved in regulation of ColE1 plasmid DNA replication. The trans-activation response element (TAR), a hairpin and bulge at the 5' end of the untranslated leader region of the human immunodeficiency virus 1 mRNA, enhances the transcription of the virus and is necessary for viral replication. Gel electrophoresis and absorbance melting curves indicate that a synthesized RNA hairpin (Tar*-16) with a loop sequence complementary to the TAR loop sequence (CUGGGA) associates specifically with a 16-nucleotide TAR hairpin (Tar-16) to form a stable complex. RNase T1 probing indicates that the three guanines in the Tar-16 loop become inaccessible in the complex. NMR imino proton spectra reveal that 5 base pairs are formed between the two hairpin loops (Tar-16 and Tar*-16); only the adenine at the 3' terminus of the TAR loop does not form a base pair with the 5'-terminal uracil of the complementary loop. A 14-nucleotide hairpin [CCUA(UCCCAG)UAGG] with a loop sequence complementary to the TAR loop is conserved within the gag gene of human immunodeficiency virus 1. A synthesized RNA hairpin corresponding to this conserved sequence also binds to the Tar-16 hairpin with high affinity. It is possible that the same RNA loop-loop interaction occurs during the viral life cycle.

Base Sequence↗
PubMed1993

Nucleoside triples from the group I intron.

Oligonucleotides modeled on a proposed base-triple domain in the P4/P6 region of the self-splicing group I intron have been characterized by NMR. The NMR data indicate that single-stranded nucleotides in this domain are in the minor groove of an adjacent helix within hydrogen bonding distance of 2'-hydroxyl groups in an interaction we term a nucleoside triple. Oligonucleotides containing the two most frequently occurring sequences among group I introns in the P4/P6 region formed nucleoside triples in the minor groove, whereas oligonucleotides containing sequences which are not conserved did not form triples. Surprisingly, the structures of the nucleoside triples in the oligonucleotides containing the two most frequently occurring sequences are different. If this difference were maintained in the context of the whole intron, it would suggest that the triples are not directly involved in catalysis, but rather that the nucleoside triples function by aligning the helical domains within the catalytic core of the intron.

Base Sequence↗
PubMed1993

An NMR study of the HIV-1 TAR element hairpin.

The TAR hairpin is an important part of the 5' long terminal repeat of HIV-1 and appears to be recognized by a cellular protein. A 14-base model of the native TAR hairpin 5'-GAGC[CUGGGA]-GCUC-3' (loop bases in square brackets) has been studied by proton, phosphorus, and natural abundance carbon NMR; these results are compared to other published NMR studies of the TAR hairpin. Assignments of all nonexchangeable protons and of all the stem-exchangeable protons have been made, as well as all phosphorus and many carbon resonances. Large J1'2' and J3'4' proton-proton coupling in the C5, G8, and G9 sugars indicate an equilibrium between C2'- and C3'-endo forms; these data show a dynamic loop structure. We see three broad imino resonances that have not been reported before; these resonances are in the right region for unbonded loop imino protons. These peaks suggest the protons are protected from fast exchange with the solvent by the structure of the hairpin loop. Simulated annealing and molecular dynamics with 148 distance constraints, 11 hydrogen bonds, and 84 torsion angle constraints showed a wide variety of structures. Certain trends are evident, such as continuation of the A-form helix on the 3' side of the hairpin loop. The ensemble of calculated structures agree with most chemical modification data.

Base Sequence↗
PubMed1993

The solution structure of a d[C(TTCG)G] DNA hairpin and comparison to the unusually stable RNA analogue.

The solution structure of the DNA analogue of the unusually stable r[C(UUCG)G] RNA hairpin, 5'-d[GGA-C(TTCG)GTCC]-3', has been determined by NMR spectroscopy, and its structure has been compared to that of the RNA molecule. The RNA molecule is compact and rigid with a highly structured loop. However, the DNA molecule is much less structured. The DNA hairpin contains a B-form stem of four base pairs. The terminal base pair frays, and the 3'-terminal nucleotides, C11 and C12, are in equilibrium between 2'-endo and 3'-endo conformations. Unlike the RNA loop, the DNA loop contains no syn nucleotides, and there is no evidence for base-base or base-phosphate hydrogen bonding in the loop. The loop is flexible, and reveals no specific internucleotide interactions.

DNA↗
PubMed1993

Structure of a small RNA hairpin.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.

Base Sequence↗
PubMed1993

The conformation of loop E of eukaryotic 5S ribosomal RNA.

The solution structure of a 27-nucleotide duplex, including the internal loop E from Xenopus laevis 5S ribosomal RNA, has been studied by two-dimensional NMR spectroscopy, followed by restrained molecular dynamics. The highly conserved internal loop closes to form a G.A base pair and a reverse-Hoogsteen A.U base pair. Extensive interstrand stacking between these uncommon base pairs provides a structural explanation for an interstrand ultraviolet-induced cross-link. A guanosine residue is bulged into the major groove and may form a base-triple with the adjacent reverse-Hoogsteen A.U pair. The structure of the less highly-conserved portion of the loop is less well-defined by the NMR data. A single-nucleotide deletion mutant has a very different, open conformation without mismatched base pairs [Varani, G., Wimberly, B., & Tinoco, I. Jr. (1989) Biochemistry 28, 7760-7772]. The implications of the structure for binding of the transcription factor TFIIIA and the cytotoxin alpha-sarcin are discussed.

Animals↗
PubMed1992

A base-triple structural domain in RNA.

An oligonucleotide modeled on a proposed base-triple domain of the Tetrahymena group I intron has been characterized by NMR. The oligonucleotide contains two double-helix regions with adjacent single-stranded nucleotides. The NMR data show that the two helices stack coaxially, although the rotation between the two helices is approximately twice as large as the rotation between normal base pairs. The rotation between the two helices allows the single-stranded nucleotides to form U.U.G and A.G.C base triples in the minor groove. The A.G.C base triple contains a hydrogen bond between the adenine N1 and a 2'-hydroxyl in the minor groove of the G.C pair. A similar hydrogen bond between an adenine and a 2'-hydroxyl in transfer RNA suggests that this could be a recurring tertiary interaction in RNA.

Animals↗
PubMed1992

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.

Base Sequence↗
PubMed1992

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.

Circular Dichroism↗
PubMed1991

A thermodynamic study of unusually stable RNA and DNA hairpins.

About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.

Base Sequence↗