Tertiary Base Pair Research Articles

Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA with its cognate synthetase

In vitro transcription by T7 RNA polymerase was used to prepare 32 different mutations in the 21 nucleotides that participate in the 9 tertiary base pairs or triples of yeast tRNAPhe. The mutations were designed either to disrupt the tertiary interaction or to change the sequence without disrupting the structure by transplanting tertiary interactions present in other tRNAs. Steady-state aminoacylation kinetics with purified yeast phenylalanyl synthetase revealed little change in reaction rate as long as a tertiary interaction was maintained. This suggests that the tertiary nucleotides only contribute to the folding of tRNAPhe and do not participate directly in sequence-specific interaction with the synthetase.

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy.

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Solution conformations of B. subtilis ribosomal 5S RNA: A calorimetric study

AbstractThe unfolding of B. subtilis 5S RNA is examined by direct calorimetric measurement in the presence of various concentrations of Na+ and Mg2+. The composite differential scanning calorimetry (DSC) curve is analyzed into 3–5 individual two‐state melting transitions. In the absence of added Na+ or Mg2+, the 5S RNA segments melt together at Tm = 40°C. Addition of Na+ stabilizes the molecular structure (Tm = 56°C) and widens the melting temperature range, so that up to five component transitions are observed. Addition of Mg2+ alone produces a very stable structure (Tm = 75°C) with highly cooperative melting. Finally, addition of both Na+ and Mg2+ produces the highest stability (Tm = 76°C). The results are interpreted according to hypothetical secondary and tertiary base‐pairing schemes. The conformational changes demonstrated here may facilitate the movement of the protein synthesis machinery during RNA translation.

Imino proton NMR assignments and ion‐binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.

Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum.

A comparison of imino proton NMR spectra of yeast tRNAPhe recorded at various solution conditions indicates, that polyamines have a limited effect on the structure of this tRNA molecule. Polyamines are found to catalyse the solvent exchange of several imino protons in yeast tRNAPhe not only of non hydrogen bonded imino protons, but also of imino protons of the GU and of some AU and tertiary base pairs. It is concluded that at low levels of catalysing components the exchange rates of the latter protons are not determined by the base pair lifetime. In the presence of high levels of spermidine the solvent exchange rates of imino protons of several base pairs in the molecule were assessed as a function of the temperature. Apparent activation energies derived from these rates were found to be less than 80 kJ/mol, which is indicative for (transient) independent opening of the corresponding base pairs. In the acceptor helix the GU base pair acts as a dynamic dislocation. The AU base pairs at one side of the GU base pair exhibit faster transient opening than the GC base pairs on the other side of this wobble pair. The base pairs m2GC10 and GC11 from the D stem and GC28 from the anticodon stem show relatively slow opening up to high temperatures. Model studies suggest that 1-methyladenosine, an element of tRNA itself, catalyses imino proton solvent exchange in a way similar to polyamines.

Nuclear magnetic resonance studies on yeast tRNAPhe. III. Assignments of the iminoproton resonances of the tertiary structure by means of nuclear Overhauser effect experiments at 500 MHz.

Resonances of the water exchangeable iminoprotons of the tertiary structure of yeast tRNAPhe were studied by experiments involving Nuclear Overhauser Effects (NOE's). Direct NOE evidence is presented for the assignment of all resonances of iminoprotons participating in tertiary basepairing (except that of G19C56 which was assigned by an elimination procedure). The present results in conjunction with our previous assignment of secondary iminoprotons constitute for the first time a complete spectral assignment of all iminoprotons participating in basepairing in yeast tRNAPhe. In addition we have been able to assign the non(internally) hydrogen bonded N1 proton of psi 55 as well as the N3 proton of this residue, which is one of the two iminoprotons hydrogen bonded to a phosphate group according to X-ray results. No evidence could be obtained for the existence in solution of the other iminoproton-phosphate interaction: that between U33 N3H and P36 located in the anticodon loop. Remarkable is the assignment of a resonance at 12.4 - 12.5 ppm to the iminoproton of the tertiary basepair T54m1A58. The resonance positions obtained for the iminoprotons of G18 (9.8 ppm) and m2(2)G26 (10.4 ppm) are surprisingly far upfield considering that these protons are involved in hydrogen bonds according to X-ray diffraction results. As far as reported by changes in chemical shifts of iminoproton resonances the main structural event induced by Mg++ ions takes place near the tertiary interactions U8A14 and G22m7G46.

Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid

The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.

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.

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 2. Species containing a large variable loop

The number of base pairs in the solution structure of several class III D3VN tRNA species from E. coli has been determined by analyzing the number of low-field (-15 to -11 ppm) proton resonances in their nuclear magnetic resonance spectra at 360 MHz. Contrary to previous reports indicating the absence of tertiary resonances, all the spectra exhibit the expected number of secondary base pair resonances plus approximately ten extra resonances derived from tertiary base pairs in the three-dimensional folding of these molecules. The possible origins of some of these tertiary resonances are discussed; none of the spectra exhibits the characteristic resonance of the 8-14 tertiary base pair seen in class I D4V5 tRNA spectra.

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.

An NMR study of the exchange rates for protons involved in the secondary and tertiary structure of yeast tRNAphe

Solvent exchange rates of all the protons of yeast tRNAphe resonating in the lowfield NMR region (-11 to-15 ppm from DSS) have been measured by saturation-recovery long-pulse Fourier transform NMR. All these protons in yeast tRNAphe are in the fast exchange limit with H2O relative to their intrinsic longitudinal relaxation processes. Most rates show very little temperature dependence; however, tertiary base pair protons are preferentially destabilized in the absence of Mg++ at higher temperatures. The measured exchange rates are between 2 and 125 sec-1 for a temperature range from 10 degrees C to 45 degrees C and MgCl2 concentrations between 0 and 15 mM.

Structlre of transfer RNA molecules containing the long variable loop.

A structure is proposed for the type II tRNA molecules containing the long variable loop and the tertiary base interactions here are compared with type I tRNAs having the short variable loop. The type II tRNAs are similar to the type I tRNAs in their tertiary base pairing interactions but differ from them generally by not having the tertiary base triples. The long variable loop, which is comprised of a helical stem and a loop at the end of it, emerges from the deep groove side of the dihydrouridine helix, and is tilted roughly 30 degrees to the plane formed by the amino acid-pseudo-uridine and anticodon-dihydrouridine helices found in yeast tRNAPhe. The fact that many of the type I tRNAs also lack the full compliment of base triples suggests that the tertiary base pairs may alone suffice to sustain the tRNA fold required for its biological function. The base triples and the variable loop appear to have little functional significance. The base type at position 9 is correlated with the number of base triples and G-C base pairs in the dihydrouridine stem.

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)

Demonstration and origin of six tertiary base pair resonances in the NMR spectrum of E. coli tRNA1Val.

The 360-MHz NMR spectrum of E. coli tRNAVal1 reveals 26 resonances, six of which are derived from tertiary base pairs. The origin of these tertiary resonances and their approximate positions in the spectrum are discussed in relation to the crystal structure of tRNA.

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+.