Determining the polarity of the map of crosslinked interactions in Escherichia coli 16 S ribosomal RNA.

Hydroxymethyltrimethylpsoralen crosslinked 16 S rRNA from Escherichia coli has been R loop hybridized to two plasmid DNAs containing different sections of the 16 S ribosomal gene. It is possible to identify crosslinked features in the part of the RNA that is not complementary to the DNA. Crosslinked features can be aligned into a relative map of interactions. Crosslinked loops that correspond to features located, originally arbitrarily, in the left part of this map are seen in the 5′ half of the 16 S rRNA in one hybrid and loops that correspond to features in the right part of the map are seen in the 3′ two-thirds of the 16 S rRNA in the other hybrid. These results confirm the relative orientations of the crosslinked loops and establish that the left end of the map corresponds to the 5′ end of the molecule.     

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologuous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A→G and C→T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TψC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

The structure of yeast tRNAAsp. A model for tRNA interacting with messenger RNA

Abstract

The anticodon of yeast tRNA^Asp, GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA^Asp molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA^Phe. In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA^Asp T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA^Phe. This variation is a consequence of the anticodon-anticodon base pairing which rigidities the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA^Asp substantiate such a correlation.     

Visualization and mapping of late nuclear adenovirus RNA

Nuclei of KB cells harvested at late stages of productive infection with adenovirus type 2 (Ad2) harbor RNA molecules which measure up to 13 μm in length, as determined by electron microscopy of denatured RNA. While some of the molecules display features of secondary structure that are characteristic for precursor rRNA, our interest was in those showing almost no intramolecular folding. When hybridized to double-stranded viral DNA under conditions which favor RNA:DNA duplex formation, nuclear AD2 RNA displaces the homologous DNA region and generates R loop structures whose size is proportional to the length of the hybridizing RNA. Slowly sedimenting RNA forms small R loops, whereas RNA of high sedimentation velocity generates loops that span a large proportion of the DNA length. Using SV40 sequences within Ad2⁺ND₄ hybrid DNA as a position marker, we oriented many of the R loops on the conventional Ad2 map. Our analysis was restricted to the most abundant sequences of late Ad2 nuclear RNA participating in R loop formation. A small but significant proportion of large RNA generates loops between map positions 0.3 and 0.9. The much more frequent RNA of intermediate size (although larger than mRNA) hybridizes with midpoints near map positions 0.55 and 0.88 — that is, near the gene locations for hexon and fiber. Our findings are compatible with the idea that the nuclear RNAs visualized in this study are intermediates in a processing pathway leading to mature forms of late Ad2 mRNA.     

Effect of photochemical crosslink S4U(8)-C(13) on suppressor activity of su+ tRNATrp from Escherichia coli.

Readthrough in vitro of the Qβ coat protein terminator codon UGA has been used as an assay for suppression by UGA-suppressor tRNA^Trp. When the tRNA is covalently crosslinked between 4-thiouracil(8) and cytosine(13) by irradiation at 334 nm, it is found that UGA suppression by this assay is reduced to the low level characteristic of the wild type tRNA^Trp. In contrast, crosslinking has little effect on incorporation of tryptophan in response to UGG codons. Thus, incorporation of tryptophan during translation of R17 messenger RNA is unaffected by photochemical crosslinking. Furthermore, dilution experiments using R17 mRNA in which tryptophan incorporation is dependent on precharged suppressor Trp-tRNA show that the crosslinked species competes well with non-irradiated tRNA. These results further emphasize the influence on tRNA-ribosome interactions of the region in tRNA around the dihydrouridine arm, where the mutation, in the suppressor is found and the photochemical crosslink is introduced.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Conformational changes in the thiouridine region of Escherichia coli transfer RNA as assessed by photochemically induced crosslinking: Effect of a single base change in the “extra” loop of formylmethionine tRNA and of denaturation of tryptophan tRNA

Photochemical crosslinking studies on two formylmethionine tRNAs of Escherichia coli are consistent with the hypothesis that the role of 7-methylguanosine is to stabilize a tertiary structure of tRNA in which the “extra” loop is folded over so as to be close to the 4-thiouridine region of the molecule. In tRNA^{fmet 3}, which differs from tRNA^{fmet 1} only by substitution of an adenosine for the 7-methylguanosine in the “extra” loop, crosslinking was virtually abolished when the tRNA was placed in 40 mm Na⁺, whereas tRNA^{fmet 1} in 40 mm Na⁺ was crosslinked to 95% of the maximum extent observed for both tRNAs in Mg²⁺. Even in Mg²⁺, a difference in structure between the two tRNAs could be detected by means of a two-fold decrease in the rate of crosslinking in tRNA^{fmet 3} as compared to tRNA^{fmet 1}. Comparison of crosslinking in the native and metastable denatured forms of tRNA^Trp of E. coli revealed that these structures also differ with respect to the orientation and/or distance between 4-thiouridine (8) and cytidine (13), since denaturation abolished crosslinking. However, separation of these two residues is not obligatory for denaturation, since crosslinked tRNA^Trp could still be denatured. A 30% difference in fluorescence between the native and denatured forms of crosslinked-reduced tRNA^Trp infers an increase in hydrophilicity in the 4-thiouridine region upon denaturation.     

Salt dependence of nucleic acid hairpin stability

Single-stranded junctions/loops are frequently occurring structural motifs in nucleic acid structures. Due to the polyanionic nature of the nucleic acid backbone, metal ions play a crucial role in the loop stability. Here we use the tightly bound ion theory, which can account for the possible ion correlation and ensemble (fluctuation) effects, to predict the ion-dependence of loop and stem-loop (hairpin) free energies. The predicted loop free energy is a function of the loop length, the loop end-to-end distance, and the ion (Na⁺ and Mg²⁺ in this study) concentrations. Based on the statistical mechanical calculations, we derive a set of empirical formulas for the loop thermodynamic parameters as functions of Na⁺ and Mg²⁺ concentrations. For three specific types of loops, namely, hairpin, bulge, and internal loops, the predicted free energies agree with the experimental data. Further applications of these empirical formulas to RNA and DNA hairpin stability lead to good agreements with the available experimental data. Our results indicate that the ion-dependent loop stability makes significant contribution to the overall ion-dependence of the hairpin stability.     

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.     

Evolutionary implications of error amplification in the self-replicating and protein-synthesizing machinery

Summary Evolutionary constraints operating on animal mitochondrial tRNA were estimated to be reduced to about 1/30 of those that apply to cytoplasmic tRNA. In the nuclear-cytoplasmic system, an effect of a mutation tRNA is likely to be amplified through positive feedback loops consisting of DNA polymerases, RNA polymerases, ribosomal proteins, aminoacyl-tRNA synthetases, tRNA processing enzymes, and others. This amplification phenomenon is called an error cascade and the loops that cause it are called error loops. The freedom of evolutionary change of cytoplasmic tRNA is expected to be severely restricted to avoid the error cascade. In fact, cytoplasmic tRNA is highly conserved during evolution. On the other hand, in the animal mitochondrial system, all of the proteins involved in error loops are coded for in the nuclear genome and imported from the cytoplasm, and accordingly the system is free from the error cascade. The difference in constraints operating on animal tRNA between cytoplasm and mitochondria is attributed to the presence or absence of error loops. It is shown that the constraints on mitochondrial tRNA in fungi are not as relaxed as those in animals. This observation is attributed to the presence of an error loop in fungal mitochondria, since at least one protein of the mitochondrial ribosome is coded for in the mitochondrial genome of fungi. The evolutionary rates of proteins involved in the processing of genetic information are discussed in relation to the error cascade.A preliminary version of this paper was presented at the International tRNA Workshop (Hakone, Japan, March 1983) and at the Second International Colloquium on Endocytobiology (Tübingen, FRG, April 1983)     

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.Abbreviations The hairpin oligonucleotides are indicated by hp hairpin followed by the loop sequence, starting at the 5'-end, in parenthesis; d for deoxy is omitted for clarity     

Higher-order structure of bovine mitochondrial tRNA(Phe) lacking the 'conserved' GG and T psi CG sequences as inferred by enzymatic and chemical probing.

Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T psi YCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNA(Phe) genes from various organisms, it was inferred that bovine mt tRNA(Phe) lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNA(Phe) to preserve its L-shaped higher-order structure.     

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.     

Defective i6A37 Modification of Mitochondrial and Cytosolic tRNAs Results from Pathogenic Mutations in TRIT1 and Its Substrate tRNA

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i⁶A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i⁶A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i⁶A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i⁶A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNA^Ser(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i⁶A37 modification. These data demonstrate that deficiencies of i⁶A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.     

Dynamics of the active site loops in catalyzing aminoacylation reaction in seryl and histidyl tRNA synthetases

Aminoacylation reaction is the first step of protein biosynthesis. The catalytic reorganization at the active site of aminoacyl tRNA synthetases (aaRSs) is driven by the loop motions. There remain lacunae of understanding concerning the catalytic loop dynamics in aaRSs. We analyzed the functional loop dynamics in seryl tRNA synthetase from Methanopyrus kandleri (^mkSerRS) and histidyl tRNA synthetases from Thermus thermophilus (^ttHisRS), respectively, using molecular dynamics. Results confirm that the motif 2 loop and other active site loops are flexible spots within the catalytic domain. Catalytic residues of the loops form a network of interaction with the substrates to form a reactive state. The loops undergo transitions between closed state and open state and the relaxation of the constituent residues occurs in femtosecond to nanosecond time scale. Order parameters are higher for constituent catalytic residues which form a specific network of interaction with the substrates to form a reactive state compared to the Gly residues within the loop. The development of interaction is supported from mutation studies where the catalytic domain with mutated loop exhibits unfavorable binding energy with the substrates. During the open-close motion of the loops, the catalytic residues make relaxation by ultrafast librational motion as well as fast diffusive motion and subsequently relax rather slowly via slower diffusive motion. The Gly residues act as a hinge to facilitate the loop closing and opening by their faster relaxation behavior. The role of bound water is analyzed by comparing implicit solvent-based and explicit solvent-based simulations. Loops fail to form catalytically competent geometry in absence of water. The present result, for the first time reveals the nature of the active site loop dynamics in aaRS and their influence on catalysis.     

R‐loop induced stress response by second (p)ppGpp synthetase in Mycobacterium smegmatis: functional and domain interdependence

Persistent R‐loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R‐loops. MS_RHII‐RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R‐loops in Escherichia coli exposed to UV stress. MS_RHII‐RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R‐loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R‐loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R‐loop‐induced stress response.     

Studies on the ribothymidine content of specific rat and human tRNAs: a postulated role for 5-methyl cytosine in the regulation of ribothymidine biosynthesis.

Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNA_i^Met); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNA^Ser, tRNA^Trp, and tRNA^Lys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNA^Phe, tRNA₁^Gly, tRNA₂^Gly); Class D contains tRNA which totally lacks ribothymidine (tRNA^Val). Only those tRNAs in Class C are acceptable substrates for $\text{E.}\text{coli}$ uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis.