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.     

Structural behavior of RNA-binding proteins in free and complex states: ribosomal protein L25 and 5S RNA from Escherichia coli

A novel approach has been proposed to evaluate the steadiness of polar clusters containing RNA-binding sites on the protein surface. The degree of clusterization of RNA-binding polar residues can be a measure of the steadiness of corresponding polar clusters. Ribosomal protein L25 from E. coli forms a complex with a fragment of 5S rRNA by means of two binding sites S1 and S2. We have examined cluster distribution of RNA-contacting polar residues on the protein surface by using the data of two states: complex state (in crystal and solution) and free state (in solution). For the crystal, the extent of clusterization of binding sites S1 and S2 are estimated to be 74.1 and 100%, respectively. For the free state in solution, the degrees of clusterization of these two sites are 22.8 and 68.6%, respectively. Thus, we have obtained a steadiness quantitative measure of two different types of protein sites for binding to RNA: one for the already existing protein binding site, and the other for the RNA-induced protein binding site. It was shown that definite variations of the protein structure in crystal and in solution can be of significant functional meaning. The result could be applied to the structural behavior of numerous protein complexes with double-stranded RNA and DNA.     

Imino proton exchange and base-pair kinetics in RNA duplexes

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton, we have measured the base-pair lifetimes and the dissociation constants of six RNA duplexes: [r(CGCGAUCGCG)](2), [r(CGCGAAUUCGCG)](2), [r(CCUUUCGAAAGG)](2), [r(CGCACGUGCG)](2), [r(GGU(8)CC).r(GGA(8)CC)], and [poly(rA).poly(rU)], and we compare them with those of their DNA homologues. As predicted by a two-state (closed/open) model of the pair, the imino proton exchange times decrease linearly vs. the inverse of catalyst concentration. As in DNA duplexes, base pairs open one at a time, and the kinetics is in most cases insensitive to the nature of the adjacent residues. The lifetime of the r(G.C) pairs, 40 to 50 ms, is longer than that of the equivalent in the corresponding oligodeoxynucleotides, and the dissociation constants, about 10(-)(7), are slightly smaller. The r(A.U) opening and closing rates are much larger than those of the d(A.T) pairs, but the stabilities are comparable.     

Escherichia coli ribosomal 5S RNA-protein L25 nucleoprotein complex: effects of RNA binding on the protein structure and the nature of the interaction

The complexes of three variants of Escherichia coli 5S RNA with ribosomal protein L25 have been studied by high-field proton nuclear magnetic resonance. A spectroscopic method is demonstrated to help distinguish the macromolecular sources of proton resonances in nucleoprotein complexes. The effects of L25 binding on the three RNAs tested were small; the presence of the L25 did not strongly influence the conformation of the RNA. The interaction of L25 with 5S RNA produced modest, but distinctive, alterations in the protein spectrum, in both the aromatic region and the upfield spectrum. As judged by these changes, the mechanism of binding was the same in all three cases. The changes seen in the spectrum of L25 indicate that its conformation is not altered in a major way upon RNA binding. Arginine residues appear to be involved in the binding mechanism. Intercalation of L25 aromatic residues with RNA bases does not appear to play a role in the interaction.     

Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA.

The topography of Escherichia coli 5S RNA has been examined in the presence of ribosomal proteins L5, L18 and L25 and their different combinations, by comparing the kethoxal modification characteristics of the various RNA-protein complexes with those of the free A-conformer of 5S RNA (Noller &; Garrett, 1979, accompanying paper).Two of the four most reactive guanines, G₁₃ and G₄₁, are unaffected by the protein, in accord with the finding that these are the only two guanines that are accessible in the 50S subunit (Noller &; Herr, 1974). The other two very reactive guanines, G₂₄ and G₆₉, are strongly protected by protein L18, either in the presence or absence of proteins L5 and L25. Protein binding studies with kethoxal-modified 5S RNA provide evidence that one or both of these two guanines are directly involved in the protein-RNA interactions, and this conclusion is supported by the occurrence of guanines in these two positions in all the other sequenced prokaryotic 5S RNAs.The group of less reactive guanines, G₁₆, G₂₃, G₄₄, G₈₆ and G₁₀₇, are protected to some extent by each of the proteins L5, L18 and L25; the strongest effect is with L18. We suggest that this is attributable to a small increase in the conformational homogeneity of the 5S RNA and that L18, in particular, induces some tightening of the RNA structure.Only one guanine, G₆₉, is rendered more accessible by the proteins. This effect is produced by protein L25, which is known to cause some destructuring of the 5S RNA (Bear et al., 1977). There was no other evidence for any destructuring of the 5S RNA. In particular, the sequence 72 to 83, which is complementary to a sequence in 23S RNA (Herr &; Noller, 1975), is not modified. However, in contrast to an earlier report (Erdmann et al., 1973), the conserved sequence G₄₄-A-A-C, which has been implicated in tRNA binding, was not rendered more accessible by the proteins.     

Escherichia coli 23S ribosomal RNA truncated at its 5'' terminus.

In a strain of E. coli deficient in RNase III (ABL1), 23S rRNA has been shown to be present in incompletely processed form with extra nucleotides at both the 5' and 3' ends (King et al., 1984, Proc. Natl. Acad. Sci. U.S. 81, 185-188). RNA molecules with four different termini at the 5' end are observed in vivo, and are all found in polysomes. The shortest of these ("C3") is four nucleotides shorter than the accepted mature terminus. In growing cells of both wild-type and mutant strains up to 10% of the 23S rRNA chains contain the 5' C3 terminus. In stationary phase cells, the proportion of C3 termini remains the same in the wild-type cells; but C3 becomes the dominant terminus in the mutant. Species C3 is also one of the 5' termini of 23S rRNA generated in vitro from larger precursors by the action of purified RNase III. We therefore suggest that some form of RNase III may still exist in the mutant; and since no cleavage is detectable at any other RNase III-specific site, the remaining enzyme would have a particular affinity for the C3 cleavage site, especially in stationary phase cells. We raise the question whether the C3 terminus has a special role in cellular metabolism.     

Heterogeneity of 5S RNA in Escherichia coli

Summary From finger-print analysis of 5S RNA isolated from 4 different Escherichia coli strains, heterogeneity of this RNA species is shown. Results point to redundancy for 5S RNA cistrons.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Evaluation of base-pairing schemes for E. coli 5S RNA by 400 MHz proton nuclear magnetic resonance spectroscopy

The number of base pairs in the denatured “B” form of $\text{E. coli}$ 5S RNA has been determined directly from 400 MHz high resolution proton nuclear magnetic resonance spectroscopy. The experimental NMR spectrum from ?11.6 to ?14.5 ppm from a sodium 2,2-dimethyl-2-silapentane sulfonate reference can be simulated by a theoretical spectrum consisting of 33 Lorentzian lines of equal width (corresponding to 33 base pairs) at 26°C. This result is inconsistent with previously proposed secondary structures of 17 and 23 base pairs, but is readily adapted to the Luoma-Marshall cloverleaf secondary structure.     

Further mapping of 5S RNA cistrons in Escherichia coli

Summary Fine mapping data for four 5S RNA cistrons in E. coli are presented.     

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.     

Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli

The imino proton spectra of several mutants of the 5S RNA of Escherichia coli are compared with that of the wild type. Three of the variants discussed are point mutations, and the fourth is a deletion mutant lacking bases 11-69 of the parent sequence, all obtained by site-directed mutagenesis techniques. The spectroscopic effects of mutation are limited in all cases, and the differences between normal and mutant spectra can be used to make or confirm the assignments of resonances. Several new assignments in the 5S spectrum are reported. Spectroscopic differences due to sequence differences permit the products of single genes within the 5S gene family to be distinguished and their fates followed by NMR.