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.     

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.     

The role of the basic N-terminal region of protein L18 in 5S RNA-23S RNA complex formation.

Of the three proteins, L5, L18 and L25, which bind to 5S RNA, the former two effect the interaction of 5S RNA with 23S RNA. We have used trypsin as a probe to investigate the roles of the proteins in this RNA-RNA assembly, with the following results: (1) In complexes with 5S RNA, the highly basic N-terminal region of L18 is accessible to trypsin. This accessibility is unaffected by L25. However, its presence is essential for stimulating L5 binding. (2) In 5S RNA-protein-23S RNA complexes proteins L5 and L18 are both strongly resistant to proteolysis. (3) No 5S RNA-23S RNA complex formation occurs in the presence of L5 and the C-terminal L18 fragment. Two possible models for the mechanism of RNA-RNA assembly are proposed.     

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).     

Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 RNA and specific 50 S subunit proteins

Pancreatic RNase partial digests of ³²P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed.     

Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S., & Garrett, R. A. (1981) Biochemistry 20, 7301--7307], reveal an extensive interaction site for protein L18 and a more localized one for L25. Generally comparable results, with a few important differences, were obtained in a study of the binding sites of the two E. coli proteins on Bacillus stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution experiments were performed for both RNAs. The effects of the bound proteins on the ribonuclease digestion of the RNAs could generally be correlated with the results obtained with the E. coli proteins L18 and L25, although there was evidence for an additional protein-induced conformational change in the B. stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5.     

Structural analyses of E. coli 5S RNA fragments, their associates and complexes with proteins L18 and L25.

The structure of Escherichia coli 5S RNA fragments 1-41 and 42-120 has been studied by the read-off gel sequencing technique using S1 nuclease and cobra venom RNase as probes. Comparison of the digestion patterns with those of reassociated and intact 5S RNA suggests that the structure of both fragments is very similar to that of the corresponding regions in the intact molecule. Six different fragments obtained by partial digestion with T1 RNase and S1 nuclease have been used for reconstitution of 5S RNA, its certain structural regions and complexes with ribosomal proteins L18 and L25 recognizes the double-helix consisting of nucleotides 79-97 (i.e. prokaryotic stem), whereas a loop-region around position 40 (possible positions 39-47) is involved in the interaction with protein L18.     

Exploration of the L18 binding site on 5S RNA by deletion mutagenesis.

Several deletion variants of E. coli 5S RNA have been constructed and produced either in vivo or in vitro using T7 RNA Polymerase. Their structures and ribosomal protein L18 binding properties have been examined. All of them are similar to wild-type 5S RNA in their helix II-III regions, where L18 binds [Huber, P.W. and Wool, I.G. (1984) Proc. Natl. Acad. Sci. (USA) 81, 322-326; Douthwaite, S., Christensen, A., and Garrett, R.A. (1982) Biochemistry 21, 2313-2320.], by NMR criteria. However, none of the molecules examined that lack the helix IV-helix V stem bind L18 efficiently, even though that portion of 5S RNA is outside the L18 footprint. The L18 binding site is clearly more than a simple hairpin loop.     

Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site.

A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA. It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent. No smaller resistant fragments were produced. 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association. Under milder trypsin digestion conditions three resistant fragments were produced from the free protein. The largest corresponded to that isolated from the complex. The smaller ones were trimmed slightly further at both N- and C-terminal ends. These smaller fragments did not reassociate with 5S RNA. It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction. The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA.     

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25.

An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees. Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18.     

Incorporation of 5S RNA into 16S-23S RNA complex

5S RNA as such is not incorporated into 16S-23S RNA complex formed under reconstitution condition. However, the addition of 50S ribosomal proteins, L5, L18 and L25/L15 results in its incorporation in stoichiometric amount. None of the proteins added individually is capable of incorporating 5S RNA into the complex. Of the different combinations in pairs that are possible out of the four proteins, the pairs L5, L18 and L15, L18 stimulate the incorporation to some extent. Of the four possible triplets, L5, L18, L25 or L5, L15, L18 is the most efficient for maximum incorporation of 5S RNA. The presence of all the four proteins is no more effective than the combinations of the three.     

Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.     

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.     

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA

A library of random mutations in Xenopus ribosomal protein L5 was generated by error-prone PCR and used to delineate the binding domain for 5S rRNA. All but one of the amino acid substitutions that affected binding affinity are clustered in the central region of the protein. Several of the mutations are conservative substitutions of non-polar amino acid residues that are unlikely to form energetically significant contacts to the RNA. Thermal denaturation, monitored by circular dichroism (CD), indicates that L5 is not fully structured and association with 5S rRNA increases the t(m) of the protein by 16 degrees C. L5 induces changes in the CD spectrum of 5S rRNA, establishing that the complex forms by a mutual induced fit mechanism. Deuterium exchange reveals that a considerable amount of L5 is unstructured in the absence of 5S rRNA. The fluorescence emission of W266 provides evidence for structural changes in the C-terminal region of L5 upon binding to 5S rRNA; whereas, protection experiments demonstrate that the N terminus remains highly sensitive to protease digestion in the complex. Analysis of the amino acid sequence of L5 by the program PONDR predicts that the N and C-terminal regions of L5 are intrinsically disordered, but that the central region, which contains three essential tyrosine residues and other residues important for binding to 5S rRNA, is likely to be structured. Initial interaction of the protein with 5S rRNA likely occurs through this region, followed by induced folding of the C-terminal region. The persistent disorder in the N-terminal domain is possibly exploited for interactions between the L5-5S rRNA complex and other proteins.     

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.