Structural behavior of RNA-binding proteins in the free state and in complex with RNA: <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomal protein L25 and 5S rRNA

A novel approach was proposed to evaluate the steadiness of polar clusters containing the RNA-binding sites on the protein surface. The degree of clustering of RNA-binding polar residues was used as a measure of the steadiness of the corresponding polar clusters. Escherichia coli ribosomal protein L25 utilizes two binding sites, S1 and S2, to complexate with a 5S rRNA fragment. The cluster distribution of RNA-contacting polar residues on the protein surface was studied using the structural data on the complex (in crystal and in solution) and the free state (in solution). The degree of polar residue clustering in S1 and S2 in crystal was estimated at 71.4 and 100%, respectively. For the free state in solution, the degree of clustering of the two sites was 22.8 and 68.6%, respectively. Thus, the steadiness was quantitatively estimated for the RNA-binding sites of two different types, one preexisting in the protein and the other induced by the RNA structure upon complexation. The difference between the protein structures in crystal and in solution was found to be functionally significant. The results can be extrapolated to numerous complexes of proteins with double-stranded RNA and DNA.     

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 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.     

Iodination of Escherichia coli ribosomal protein L18 abolishes its 5 S RNA binding activity

Iodination of Escherichia coli ribosomal protein L18 inactivated the 5 S RNA binding activity of the protein. Complete activity loss occurred at a 4-fold molar excess of iodine to L18. Tyrosine was found to be the reactive amino acid. L18, prebound to 5 S RNA, was inactivated at a much slower rate than unbound L18. Treatment of L18 with tetranitromethane also resulted in an inactivation of the protein. However, much larger amounts of tetranitromethane, compared to iodine, were necessary to achieve inactivation (50% activity loss at a 600-fold molar excess of tetranitromethane to L18).     

Structural properties of ribosomal protein L11 from Escherichia coli

Protein L11 has been isolated from the large subunit of the E. coli ribosome under non-denaturing conditions and studied by proton magnetic resonance spectroscopy, limited proteolysis, and fluorescence and UV spectroscopy. The protein consists of two domains, a tightly-folded N-terminal part and a C-terminal half with an extended and loosely folded conformation. It is likely that the N-terminal domain is located on the surface of the subunit whereas the C-terminal part is buried within the ribosomal structure. The two tyrosines in the N-terminal region behave as solvent-exposed residues, in good agreement with iodination studies on L11 in situ. It appears probable that the central region of L11, in which the protease cleavages occur, plays an important part in structural and functional aspects.     

Alteration of 5S RNA conformation by ribosomal proteins L18 and L25.

The effects of ribosomal proteins L18, L25 and L5 on the conformation of 5S RNA have been studied by circular dichroism and temperature dependent ultraviolet absorbance. The circular dichroism spectrum of native 5S RNA is characterized in the near ultraviolet by a large positive band at 267 nm and a small negative band at 298 nm. The greatest perturbation in the spectrum was produced by protein L18 which induced a 20% increase in the 267 nm band and no change in the 298 nm band. By contrast, protein L25 caused a small decrease in both bands. No effect was observed with protein L5. Simultaneous binding of proteins L18 and L25 resulted in CD changes equivalent to the sum of their independent effects. The UV absorbance thermal denaturation profile of the 5S RNA L18 complex lacked the pre-melting behavior characteristic of 5S RNA. Protein L25 had no effect on the 5S RNA melting profile. We concluded that protein L18 increases the secondary, and possible the tertiary structure of 5S RNA, and exerts a minor stabilizing effect on its conformation while protein L25 causes a small decrease in 5S RNA secondary structure. The implications of these findings for ribosome assembly and function are discussed.     

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.     

5S ribosomal RNA is contained within a 25 S ribosomal RNA that accumulates in mutants of Escherichia coli defective in processing of ribosomal RNA.

A temperature-sensitive mutant strain of Escherichia coli defective in two RNA processing enzymes, RNase III and RNase E (rnc. rne), fails to produce normal levels of 23 S and 5 S rRNA at the non-permissive temperature. Instead, a molecule larger than 23 S is produced. This molecule, designated 25 S rRNA, can be processed in vitro to produce p5 rRNA. These findings further our understanding of the overall processing events of ribosomal RNA which take place in the bacterial cell.     

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)     

RNA sequences in ribonucleoprotein fragments of the complex formed from ribosomal 23-S RNA and ribosomal protein L24 of Escherichia coli.

Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA. By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabilised by protein L24.     

The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli

Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1. A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11. The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests. It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E. coli 23 S rRNA.     

The binding site for ribosomal protein complex L8 within 23 s ribosomal RNA of Escherichia coli

The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10. This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1. Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins. They were also subjected to nucleotide sequence analysis. The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study. Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA. It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E. coli.     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

Characterization of Escherichia coli 50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.