Thermodynamic characterization of RNA duplexes containing naturally occurring 1 x 2 nucleotide internal loops

Thermodynamic data for RNA 1 x 2 nucleotide internal loops are lacking. Thermodynamic data that are available for 1 x 2 loops, however, are for loops that rarely occur in nature. In order to identify the most frequently occurring 1 x 2 nucleotide internal loops, a database of 955 RNA secondary structures was compiled and searched. Twenty-four RNA duplexes containing the most common 1 x 2 nucleotide loops were optically melted, and the thermodynamic parameters DeltaH degrees , DeltaS degrees , DeltaG degrees 37, and TM for each duplex were determined. This data set more than doubles the number of 1 x 2 nucleotide loops previously studied. A table of experimental free energy contributions for frequently occurring 1 x 2 nucleotide loops (as opposed to a predictive model) is likely to result in better prediction of RNA secondary structure from sequence. In order to improve free energy calculations for duplexes containing 1 x 2 nucleotide loops that do not have experimental free energy contributions, the data collected here were combined with data from 21 previously studied 1 x 2 loops. Using linear regression, the entire dataset was used to derive nearest neighbor parameters that can be used to predict the thermodynamics of previously unmeasured 1 x 2 nucleotide loops. The DeltaG degrees 37,loop and DeltaH degrees loop nearest neighbor parameters derived here were compared to values that were published previously for 1 x 2 nucleotide loops but were derived from either a significantly smaller dataset of 1 x 2 nucleotide loops or from internal loops of various sizes [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912-4924]. Most of these values were found to be within experimental error, suggesting that previous approximations and assumptions associated with the derivation of those nearest neighbor parameters were valid. DeltaS degrees loop nearest neighbor parameters are also reported for 1 x 2 nucleotide loops. Both the experimental thermodynamics and the nearest neighbor parameters reported here can be used to improve secondary structure prediction from sequence.     

The nucleotide sequence of threonine transfer RNA coded by bacteriophage T4.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.     

Thermodynamic characterization of RNA 2 × 3 nucleotide internal loops

To better elucidate RNA structure-function relationships and to improve the design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2 × 3 nucleotide loops are based on a 23-member data set of 2 × 3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2 × 3 nucleotide loops that occur in nature. Seventeen of the most frequent 2 × 3 nucleotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°(37,2×3) values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912-4924] and Chen and Turner [Chen, G., and Turner, D. H. (2006) Biochemistry 45, 4025-4043], respectively. These new ΔG°(37,2×3) values can be used to update the current algorithms that predict secondary structure from sequence. To improve free energy calculations for duplexes containing 2 × 3 nucleotide loops that still do not have experimentally determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2 × 3 nucleotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of those of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2 × 3 nucleotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

Nucleotide sequence of starfish initiator tRNA.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.     

Biochemical research on oogenesis. Nucleotide sequence of initiator tRNA from oocytes and from somatic cells of Xenopus laevis.

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs. Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.     

Identification of two methionine transfer RNA genes in the maize mitochondrial genome

Two methionine transfer RNA (tRNA) genes were identified in the maize mitochondrial genome by nucleotide sequence analysis. One tRNA gene was similar in nucleotide sequence and secondary structure to the initiator methionine tRNA genes of eubacteria and higher plant chloroplast genomes. This tRNA gene also had extensive nucleotide homology (99%) with an initiator methionine tRNA gene described for the wheat mitochondrial genome. The other methionine tRNA gene sequence was distinct and more closely resembled an elongator methionine tRNA.     

Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila.

The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques. The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH. The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No. 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms. The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.     

Rapid separation of tyrosine-specific tRNA from white lupin.

During isolation of total ribonucleic acids from white lupin (Lupinus albus) and their subsequent separation by 10% polyacrylamide gel electrophoresis, a fast migrating RNA band is very well separated. The nucleotide sequence analysis of 76 nucleotide long sequence with many modified nucleosides was found to be identical with that of tyrosine specific tRNA of yellow lupin seeds (Lupinus luteus) and wheat germ (Triticum aestivum). Also this tRNA(Tyr) is identical with plant amber suppressor tRNA. The presented approach offers a very rapid method of purification of plant tRNA with UAG suppressor activity.     

Prediction of the frequencies of restriction endonuclease recognition sequences using di- and mononucleotide frequencies

The calculation of probabilities of nucleotide sequences from the frequencies of dinucleotides is described. The dinucleotide and mononucleotide frequencies used can be obtained from nearest neighbor analysis or from databank sequences. If dinucleotide and mononucleotide frequencies from nearest neighbor analysis are used, probabilities for oligonucleotides can be calculated for genomes in which there is little or no sequence data. Within a given genome, a broad range of probabilities for hexanucleotide palindromes with the same base composition is predicted and shown (14).     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

Rabbit liver tRNA1Val:I. Primary structure and unusual codon recognition.

The major valine acceptor tRNA1Val from rabbit liver was purified and its nucleotide sequence determined by in vitro [32P] - labeling with T4 phage induced polynucleotide kinase and finger-printing techniques. Its primary structure was found to be identical with the major valine tRNA from mouse myeloma cells. According to the wobble hypothesis this tRNA, which exclusively has an IAC anticodon, should decode the valine codons GUU, GUC and GUA only. However, this tRNA recognizes all four valine codons with a surprising preference for GUG. It is unknown whether this is due to the lack of A37 modification next to the 3' end of the anticodon IAC. The nature of the inosine-guanosine interaction remains to be clarified.     

A primer ribonucleic acid for initiation of in vitro Rous sarcarcoma virus deoxyribonucleic acid synthesis.

The nucleotide sequence of an RNA primer molecule for initiation of Rous sarcoma virus DNA synthesis in vitro has been determined. The sequence can be drawn in a cloverleaf structure typical of tRNAs with an anticodon for tryptophan. Aminoacylation of the molecule confirms that it is tRNA-Trp. The same sequence and aminoacylation results are obtained regardless of whether the RNA is isolated from virions or from cells of chickens, the natural host for this virus. It is the only species of tRNA-Trp that is dectected in chicked cell tRNA.     

Structural characterization of naturally occurring RNA single mismatches

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence–structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences.     

Nucleotide sequence of initiator tRNA from Mycobacterium smegmatis.

The nucleotide sequence of initiator tRNA from Mycobacterium smegmatis was determined to be pCGCGGGGUGGAGCAGCUCGGDAGCUCGCUGGGCUCAUAACCCAGAGm7GUCG CAGGU psi CGm1AAUCCUGUCCCCGCUACCAOH . The nucleotide sequence of Mycobacterium initiator tRNA was found to be the same as that of Streptomyces initiator tRNA, except that G46 and A57 were replaced by m7G46 and G57 , respectively. The striking feature of Mycobacterium initiator tRNA is the absence of ribothymidine at residue 54, and the presence of 1-methyladenosine at residue 58 which makes the sequence of this tRNA similar to that of eukaryotic initiator tRNA.