Tertiary Structure Base Pairs Research Articles

Crucial role of the C-terminal domain of Mycobacterium tuberculosis leucyl-tRNA synthetase in aminoacylation and editing

The C-terminal extension of prokaryotic leucyl-tRNA synthetase (LeuRS) has been shown to make contacts with the tertiary structure base pairs of tRNALeu as well as its long variable arm. However, the precise role of the flexibly linked LeuRS C-terminal domain (CTD) in aminoacylation and editing processes has not been clarified. In this study, we carried out aspartic acid scanning within the CTD of Mycobacterium tuberculosis LeuRS (MtbLeuRS) and studied the effects on tRNALeu-binding capacity and enzymatic activity. Several critical residues were identified to impact upon the interactions between LeuRS and tRNALeu due to their contributions in the maintenance of structural stability or a neutral interaction interface between the CTD platform and tRNALeu elbow region. Moreover, we propose Arg921 as a crucial recognition site for the tRNALeu long variable arm in aminoacylation and tRNA-dependent pre-transfer editing. We also show here the CTD flexibility conferred by Val910 in regulation of LeuRS–tRNALeu interaction. Taken together, our results suggest the structural importance of the CTD in modulating precise interactions between LeuRS and tRNALeu during the quality control of leucyl-tRNALeu synthesis. This system for the investigation of the interactions between MtbLeuRS and tRNALeu provides a platform for the development of novel antitubercular drugs.

Structural Constraints Identified with Covariation Analysis in Ribosomal RNA

Covariation analysis is used to identify those positions with similar patterns of sequence variation in an alignment of RNA sequences. These constraints on the evolution of two positions are usually associated with a base pair in a helix. While mutual information (MI) has been used to accurately predict an RNA secondary structure and a few of its tertiary interactions, early studies revealed that phylogenetic event counting methods are more sensitive and provide extra confidence in the prediction of base pairs. We developed a novel and powerful phylogenetic events counting method (PEC) for quantifying positional covariation with the Gutell lab’s new RNA Comparative Analysis Database (rCAD). The PEC and MI-based methods each identify unique base pairs, and jointly identify many other base pairs. In total, both methods in combination with an N-best and helix-extension strategy identify the maximal number of base pairs. While covariation methods have effectively and accurately predicted RNAs secondary structure, only a few tertiary structure base pairs have been identified. Analysis presented herein and at the Gutell lab’s Comparative RNA Web (CRW) Site reveal that the majority of these latter base pairs do not covary with one another. However, covariation analysis does reveal a weaker although significant covariation between sets of nucleotides that are in proximity in the three-dimensional RNA structure. This reveals that covariation analysis identifies other types of structural constraints beyond the two nucleotides that form a base pair.

Tertiary structure base pairs between D- and TpsiC-loops of Escherichia coli tRNA(Leu) play important roles in both aminoacylation and editing.

To ensure the fidelity of protein biosynthesis, aminoacyl-tRNA synthetases (aaRSs) must recognize the tRNA identity elements of their cognate tRNAs and discriminate their cognate amino acids from structurally similar ones through a proofreading (editing) reaction. For a better understanding of these processes, we investigated the role of tRNA(Leu) tertiary structure in the aminoacylation and editing reactions catalyzed by leucyl-tRNA synthetase (LeuRS). We constructed a series of Escherichia coli tRNA(Leu) mutated transcripts with alterations of the nucleotides involved in tertiary interactions. Our results revealed that any disturbance of the tertiary interaction between the tRNA(Leu) D- and TpsiC-loops affected both its aminoacylation ability and its ability to stimulate the editing reaction. Moreover, we found that the various tertiary interactions between the D- and TpsiC-loops (G18:U55, G19:C56 and U54:A58) functioned differently within the aminoacylation and editing reactions. In these two reactions, the role of base pair 19:56 was closely correlated and dependent on the hydrogen bond number. In contrast, U54:A58 was more important in aminoacylation than in editing. Taken together, our results suggest that the elbow region of tRNA formed by the tertiary interactions between the D- and TpsiC-loops affects the interactions between tRNA and aaRS effectively both in aminoacylation and in editing.

A Study of the Thermal Unfolding of Escherichia coli Phenylalanine Transfer RNA by Chemical Modification at Elevated Temperatures

Escherichia coli tRNAPhe was modified by 3 M sodium bisulphite pH 6.0 for 24 h in the temperature range 25 degrees C (x 5 degrees C) to 55 degrees C and in the absence of added magnesium ions. The sites and extents of conversion of cytidines to uridine occurring at each temperature were determined by fingerprinting. The new sites of cytidine modification found at higher reaction temperatures were assumed to arise from breakage of secondary and tertiary structure hydrogen bonds involving cytidine residues. From these data, we conclude that hydrogen bonds within the 'complex core' of the tRNA (including the base pairs G-10 . C-25, C-11 . G-24 and C-13 . G-21 within the dihydrouridine stem and the tertiary structure base pair G-15 . C-48 melt at a lower temperature than the tertiary structure hydrogen bonds between G-19 in the dihydrouridine loop and C-56 in the TpsiC loop.

Hydrogen-bonded protons in the tertiary structure of yeast tRNAPhe in solution.

Temperature-dependent lowfield proton magnetic resonance spectra of yeast tRNAPhe were recorded between 10 and 15 parts per million. Seven resonances of hydrogen-bonded protons disappeared reversibly under two sets of conditions where the selective broadening of tertiary structure resonances were predicted by temperature jump experiments. The seven resonances were assigned to the seven tertiary hydrogen bonds expected between 10 and 15 parts per million from the crystal structure of yeast tRNAPhe. Some of the non-Watson-Crick base pairs have unusual unshifted standard chemical shifts after the ring current contributions calculated from the crystal coordinates were subtracted. The differences of the chemical shifts of homologous tertiary structure base pairs in Escherichia coli tRNAfMet and yeast tRNAPhe give experimental evidence for details of the conformational differences postulated by model building on the basis of the x-ray coordinates of yeast tRNAPhe and sequence homologies.

Quantitative determination of the number of secondary and tertiary structure base pairs in transfer RNA in solution

Resonances in the low-field (11-15 ppm) nuclear magnetic resonance spectrum (NMR) of tRNA molecules arise from secondary and tertiary structure base pairs (1 resonance for each base pair) as well as tertiary structure hydrogen bonds. An accurate method for integrating the low-field spectra has been developed and applied to seven different tRNA. In the presence of high levels of magnesium (10 mM free magnesium) the number of resonances (base pairs) per molecule is typically 3-4 more than the number predicted by the cloverleaf model. These results confirm our recent proposal that, under proper conditions, most tRNA exhibit 3-4 tertiary structure interactions in solution, which are also observed in x-ray diffraction studies of yeast tRNAPhe. In addition to common resonances in the 11-15 ppm region, there are common resonances at 10.5 and 9.5 ppm. A critique of methods used to integrate the low-field spectra is given and possible sources of error are indicated. The discrepancy between our present results and previous studies, which indicated that the number of base pairs per molecule was close to the number predicted by the cloverleaf model, can be attributed partly to differences in magnesium concentration and partly to inaccuracies inherent in the integration methods used.

Letter: Quantification of secondary and tertiary structure base pairs in E. coli tRNA1Val.

Letter: Quantification of secondary and tertiary structure base pairs in E. coli tRNA1Val.

Quantification of secondary and tertiary structure base pairs in E. coli tRNA1Val (reply)

Quantification of secondary and tertiary structure base pairs in E. coli tRNA1Val (reply)

Nuclear magnetic resonance of the base-pairing structure of the native and denatured conformers of Escherichia coli transfer RNA Trp

The native and denatured forms of transfer RNA Trp from Escherichia coli have been studied by high-resolution (300 MHz) proton nuclear magnetic resonance spectroscopy. The low-field nuclear magnetic resonance spectrum indicates that there are 22 ± 2 base-pairs present in the native form, and the observed resonance positions can be accounted for in terms of 19 base-pairs from the cloverleaf secondary structure, one protected U, and two tertiary structure base-pairs including A 14-s 4U 8. The denatured conformer contains one or two less base-pairs than the native conformer, and the low-field spectrum of the denatured conformer can be accounted for in terms of a model which retains the amino acid acceptor stem, T ΨC stem, and the anticodon stem of the cloverleaf model, but which pairs some bases of the dihydrouridine stem with bases from the minor loop. The proposed model is consistent with the ease of interconversion of the native and denatured conformers, the pattern of kethoxalation of the native and denatured conformera, and the retention of a tertiary structure A 14-s 4U 8 base-pair in the denatured conformer. Alternative models of the denatured form are considered, but they do not adequately explain the observed nuclear magnetic resonance spectrum. The binding of chloroquine to the native conformer has also been studied. A comparison of the binding of ethidium bromide and chloroquine to E. coli tRNA Glu indicates that the two drugs have very similar binding properties and this suggests a possible mechanism for the chloroquine-induced renaturation of tRNA Trp.

NMR evidence for common tertiary structure base pairs in yeast and E. coli tRNA.

ABOUT ten years ago Holley1 and coworkers sequenced the first tRNA and proposed the now famous cloverleaf secondary structure. Since that time about sixty additional tRNAs have been sequenced and all fit the cloverleaf model2. This homology constitutes a strong case for the cloverleaf model and there is now compelling evidence from nuclear magnetic resonance (NMR) studies3 that the clover-leaf model does provide a correct description of the secondary structure of tRNA molecules in solution.

Tertiary structure in E. coli tRNA Arg and tRNA Val

300 MHz proton NMR has been used to demonstrate that both Escherichia coli tRNA A rg and tRNA V al 1 have a tertiary structure base pair between 4-thiouridine (s 4U) at position 8 and A at position 14. Formation of this s 4U 8-A 14 base pair leads to a very low field (14.8 ppm) resonance in the spectra of both molecules. When s 4U is converted to U the 14.8 resonance is replaced by a new resonance which appears at 14.3 ppm. The presence of the tertiary structure s 4U 8-A 14 base pair greatly constrains the folding of these tRNAs in solution.

Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution Nmr.

AbstractThe high‐resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without Mg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A14–S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg2+ the tertiary structure base pair is present only to ∼45°C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14–S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg2+.

NMR evidence for tertiary structure base pair in E. coli tRNA involving S4U8.

IN previous nuclear magnetic resonance (NMR) studies of tRNA we observed anomalously low field resonances (14.5–15.0 p.p.m. downfield from the usual standard 2,2-dimethylsilapentane-5-sulphonate (DSS)) in the spectra of many, but not all, Escherichia coli tRNA1–3 but yeast tRNA we examined never exhibited such resonances1,2. The fact that approximately 60% of the E.coli tRNA are class I and have a 4-thiouridine (S4U) residue at position 8 (refs 4 and 5), whereas yeast tRNA do not, raised the possibility that the anomalous low field resonances in E. coli tRNA might be due to ring NH protons of S4U8 in a tertiary structure base pair.