Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25

The downfield (9-15 ppm) proton spectrum of Escherichia coli 5S RNA has been examined at 500 MHz by using nuclear Overhauser methods. The data confirm the existence of the terminal and procaryotic loop helices within the molecule [Fox, G. E., & Woese, C. R. (1975) Nature (London) 256, 505-506]. Very little stable, double-helical structure is detectable in the third loop of the molecule, the one comprising bases 12-68. The downfield spectrum of 5S RNA is perturbed in a highly specific manner upon addition of protein L25 to the system. The changes seen strongly suggest that the binding site for L25 on 5S RNA includes the procaryotic loop helix, but not the terminal stem helix. Similar complexes formed between L25 and the 5S RNA fragment consisting of bases 1-11, 69-87, and 89-120 show exactly the same spectral alterations. A number of downfield resonances appear in the spectra of these complexes which have no counterparts in the free RNA, suggesting the stabilization of new RNA structures by the protein. There are some indications of protein-nucleic acid nuclear Overhauser effects.     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

NMR evidence for dynamic secondary structure in helices II and III of the RNA of Escherichia coli

A new ribonuclease A (RNase A) resistant fragment of the 5S ribonucleic acid (RNA) from Escherichia coli has been isolated and characterized. This fragment comprises helix III and most of helix II of the parent molecule, a part of the 5S RNA molecule for which several energetically equivalent secondary structures have been proposed [De Wachter, R., Chen, M.-W., & Vandenberghe, A. (1984) Eur. J. Biochem. 143, 175-182]. The imino proton spectrum of this fragment has been studied by nuclear magnetic resonance methods at 500 MHz. The data obtained are readily rationalized in terms of one of the structures proposed for this region of 5S RNA. They also suggest that upon heating, this structure is replaced by a second, different one, consistent with the view that the helix II-helix III region of 5S RNA is able to switch between alternative structures. Among the products of the nucleolytic digestion of 5S RNA is a species whose sequence indicates that RNase A can ligate RNA as well as hydrolyze it.     

Assignment of resonances in the downfield proton spectrum of Escherichia coli 5S RNA and its nucleoprotein complexes using components of a ribonuclease-resistant fragment

The downfield (9-15 ppm) proton NMR spectra of oligonucleotides derived from the ribonuclease A resistant fragment of Escherichia coli 5S RNA have been examined in aqueous solution at 500 MHz. Comparison of these spectra with those of the 5S RNA fragment and intact 5S RNA using both chemical shift and nuclear Overhauser enhancement effect criteria indicates that several aspects of 5S RNA secondary structure are also present in the structures assumed in solution by these much smaller molecules. Analysis of these spectra permits the assignment of some imino proton resonances which could not be assigned with certainty on the basis of NMR data previously obtained on intact 5S RNA or its nucleoprotein complexes. Several previous resonance assignments are confirmed. Studies on oligonucleotide components of fragment and a reconstituted fragment show that at least two conformations of the procaryotic loop exist.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

NMR study of isoleucine transfer RNA from Thermus thermophilus

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.     

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.     

Imino proton exchange in the 5S RNA of Escherichia coli and its complex with protein L25 at 490 MHz

Imino proton exchange has been examined by NMR in the 5S RNA of Escherichia coli, its principal RNase A resistant fragment, fragment 1 (bases 1-11, 69-120), and complexes between that fragment and ribosomal protein L25 by using both real-time and relaxation techniques. Fragment 1 RNA imino protons exchange at rates between 0.5 and 15 s-1 at 303 K in 5 mM cacodylate buffer, pH 7.4. In contrast with many tRNAs, intact 5S RNA contains no imino protons with exchange lifetimes as great as 1 min. Consistent with the results of Gueron and his colleagues [Leroy, J. L., Bolo, N., Figueroa, N., Plateau, P., & Gueron, M. (1985) J. Biomol. Struct. Dyn. 2,915-939; Leroy, J. L., Broseta, D., & Gueron, M. (1985) J. Mol. Biol. 184, 165-178] with tRNA, exchange in 5S RNA is catalyst-limited under conditions generally used for imino proton spectroscopy, such as those given above. Using Gueron's catalyst saturation technique, base pair opening rates have been measured for several AU and GU base pairs in fragment 1. They range from 50 to 300 s-1 at 303 K and depend on base pair type and also to some degree on context. Similar studies have been done on complexes of L25 and fragment 1. The binding of L25 to fragment 1 reduces the exchange rate of many imino protons within the region to which it binds, consistent with the hypothesis that its binding stabilizes the secondary structure of 5S RNA.     

Sequence dependence for the energetics of terminal mismatches in ribooligonucleotides

Stability increments of terminal mismatches on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 15 oligoribonucleotides containing the core sequences and various mismatches. Free energy increments for mismatches in this series range from -0.5 to -1.1 kcal/mol. These increments for mismatches on AU base pairs are smaller than those measured previously on GC base pairs [Freier, S.M., Kierzek, R., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Biochemistry 25, 3209-3213]. The terminal GU mismatches in the sequences GAUGCAUUp and UAUGCAUGp add approximately the same stability increment as the corresponding terminal AU mismatch. The stability increments for pyrimidine-pyrimidine and pyrimidine-purine mismatches can be approximated within 0.3 kcal/mol by adding the stability increments for the corresponding 3' and 5' unpaired nucleotides (dangling ends). Stability increments for purine-purine mismatches are approximated well by the stability increment for the corresponding 3' dangling end made more favorable by 0.2 kcal/mol. These approximations are used to provide a table of stability increments for all 48 possible sequences of mismatches.     

Nucleotide sequence of 7 S RNA. Homology to Alu DNA and La 4.5 S RNA

7 S RNA, a component of normal higher eukaryotic cells and several oncornaviruses, was shown to be conserved in evolution (Erikson, E., Erikson, R. L., Henry, B., and Pace, N. R. (1973) Virology 53, 40-46). Recently, 7 S RNA was shown to be partially complementary to Alu family DNA sequences (Weiner, A. (1980) Cell 22, 209-218). In the present study the nucleotide sequence of Novikoff hepatoma 7 S RNA was determined to be: (formula, see text) Comparison of 7 S RNA, Alu and B1 family DNA, and La 4.5 S RNA sequences for homologies showed that 1) one-third of 7 S RNA, mainly the 5'-end, was homologous to Alu and B1 family sequences; 2) one 300-nucleotide long Alu family sequence contained two binding sites for 7 S RNA; and 3) the 5'-ends of 7 S RNA and La 4.5 S RNA also had extensive (60%) homologies. A model for the secondary structure of 7 S RNA based on maximal base pairing and preferential nuclease cleavage sites is also presented.     

An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli

Experiments are described that complete the assignment of the imino proton NMR spectrum of the fragment 1 domain from the 5S RNA of Escherichia coli. Most of the new assignments fall in the helix V-loop E portion of the molecule (bases 70-78 and 98-106), the region most sensitive to the binding of ribosomal protein L25. The spectroscopic data are incompatible with the standard, phylogenetically derived model for 5S RNA, which makes all the base pairs possible in loop E with the sequences aligned in parallel (C70-G106, C71-G105, etc.) [see Delihas et al. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190]. Furthermore, the alternative loop E model proposed for spinach chloroplast 5S RNA by Romby et al. [(1988) Biochemistry 27, 4721-4730] does not apply to the closely homologous 5S RNA from E. coli. The 5S RNAs from E. coli and spinach chloroplasts do not have the same secondary structures in solution despite their strong sequence homologies, and neither appears to conform to the standard model for 5S RNA in the loop E region.     

Computing the partition function and sampling for saturated secondary structures of RNA, with respect to the Turner energy model.

An RNA secondary structure is saturated if no base pairs can be added without violating the definition of secondary structure. Here we describe a new algorithm, RNAsat, which for a given RNA sequence a, an integral temperature 0 相似文献   

Secondary structure of a 345-base RNA fragment covering the S8/S15 protein binding domain of Escherichia coli 16S ribosomal RNA

A technique for isolating defined fragments of a large RNA has been developed and applied to a ribosomal RNA. A section of the Escherichia coli rrnB cistron corresponding to the S8/S15 protein binding domain of 16S ribosomal RNA was cloned into a single-stranded DNA phage; after hybridization of the phage DNA with 16S RNA and digestion with T1 ribonuclease, the protected RNA was separated from the DNA under denaturing conditions to yield a 345-base RNA fragment with unique ends (bases 525-869 in the 16S sequence). The secondary structure of this fragment was determined by mapping the cleavage sites of enzymes specific for single-stranded or double-helical RNA. The fragment structure is almost identical with that proposed for the corresponding region of intact 16S RNA on the basis of phylogenetic comparisons [Woese, C. R., Gutell, R., Gupta, R., & Noller, H. (1983) Microbiol. Rev. 47, 621-669]. We conclude that this section of RNA constitutes an independently folding domain that may be studied in isolation from the rest of the 16S RNA. The structure mapping experiments have indicated several interesting features in the RNA structure. (i) The largest bulge loop in the molecule (20 bases) contains specific tertiary structure. (ii) A region of long-range secondary structure, pairing bases about 200 residues apart in the sequence, can hydrogen bond in two different mutually exclusive schemes. Both appear to exist simultaneously in the RNA fragment under our conditions. (iii) The long-range secondary structure and one adjacent helix melt between 37 and 60 degrees C in the absence of Mg2+, while the rest of the structure is quite stable.     

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.     

Characterization of the secondary structure and melting of a self-cleaved RNA hammerhead domain by 1H NMR spectroscopy.

We have completely assigned the extreme low-field ring-NH nuclear magnetic resonance spectrum of a self-cleaving RNA in the absence of magnesium ions by experiments involving sequential Overhauser enhancements between adjacent base pairs. These assignments substantiate the hammerhead secondary folding model proposed by Symons and co-workers for this class of self-cleaving RNA [Hutchins, C. J., Rathjen, P. D., Forster, A. C., & Symons, R. H. (1986) Nucleic Acids Res. 14, 3627-3640; Forster, A. C. & Symons, R. H. (1987) Cell 49, 211-220; Kneese, P., & Symons, R. H. (1987) in Viroids and Viroid-like Pathogens (Semancick, J. S., Ed.) pp 1-47, CRC Press, Boca Raton, FL]. No resonances due to tertiary base pairs could be identified in the low-field spectrum, and addition of MgCl2 to the sample did not produce additional resonances in this region of the spectrum.     

Defining the interface between the C-terminal fragment of alpha-transducin and photoactivated rhodopsin

A novel combination of experimental data and extensive computational modeling was used to explore probable protein-protein interactions between photoactivated rhodopsin (R*) and experimentally determined R*-bound structures of the C-terminal fragment of alpha-transducin (Gt(alpha)(340-350)) and its analogs. Rather than using one set of loop structures derived from the dark-adapted rhodopsin state, R* was modeled in this study using various energetically feasible sets of intracellular loop (IC loop) conformations proposed previously in another study. The R*-bound conformation of Gt(alpha)(340-350) and several analogs were modeled using experimental transferred nuclear Overhauser effect data derived upon binding R*. Gt(alpha)(340-350) and its analogs were docked to various conformations of the intracellular loops, followed by optimization of side-chain spatial positions in both R* and Gt(alpha)(340-350) to obtain low-energy complexes. Finally, the structures of each complex were subjected to energy minimization using the OPLS/GBSA force field. The resulting residue-residue contacts at the interface between R* and Gt(alpha)(340-350) were validated by comparison with available experimental data, primarily from mutational studies. Computational modeling performed for Gt(alpha)(340-350) and its analogs when bound to R* revealed a consensus of general residue-residue interactions, necessary for efficient complex formation between R* and its Gt(alpha) recognition motif.     

500-MHz proton NMR evidence for two solution structures of the common arm base-paired segment of wheat germ 5S ribosomal RNA

The base-pair protons of the common arm duplex fragment of wheat germ (Triticum aestivum) ribosomal 5S RNA have been identified and assigned by means of 500-MHz proton NMR spectroscopy. The two previously reported extra base pairs within the fragment [Li et al. (1987) Biochemistry 26, 1578-1585] are now explained by the presence of two distinct solution structures of the common arm fragment (and its corresponding base-paired segment in intact 5S rRNA). The present conclusions are supported by one- and two-dimensional proton homonuclear Overhauser enhancements in H2O and by temperature variation and Mg2+ titration of the downfield 1H NMR spectrum. The difference between the two conformers is most likely due to difference in helical tightness. Some additional amino proton resonances have also been assigned.     

Structural model for an oligonucleotide containing a bulged guanosine by NMR and energy minimization

We present three-dimensional structural models for a DNA oligomer containing a bulged guanosine based on proton NMR data and energy minimization computations. The nonexchangeable proton resonances of the duplex 5'd(GATGGGCAG).d(CTGCGCCATC) are assigned by nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy connectivities, and the NMR spectrum is compared with that of a regular 8-mer of similar sequence, 5'd(GATGGCAG).d(CTGCCATC). Experimental proton-proton distances are obtained from NOESY spectra acquired with mixing times of 100, 150, and 200 ms. A refined three-dimensional structure for the bulge-containing duplex is calculated from regular B DNA starting coordinates by using the AMBER molecular mechanics program [Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., Profeta, S., & Weiner, P. (1984) J. Am. Chem. Soc. 106, 765-784]. We compare structures obtained by building the helix in three and four base pair increments with structures obtained by direct minimization of the entire nine base sequence, with and without experimental distance constraints. The general features of all the calculated structures are very similar. The helix is of the B family, with the extra guanine stacked into the helix, and the helix axis is bent by 18-23 degrees, in agreement with gel mobility data for bulge-containing sequences [Rice, J. A. (1987) Ph.D. Thesis, Yale University].