Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli

The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities. The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier. They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions. (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur. (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III. Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre. Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region.     

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.     

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5''-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.     

Ribosome-DnaK interactions in relation to protein folding

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.     

Disruption of shape complementarity in the ribosomal protein L1–RNA contact region does not hinder specific recognition of the RNA target site

The formation of a specific and stable complex between two (macro)molecules implies complementary contact surface regions. We used ribosomal protein L1, which specifically binds a target site on 23S rRNA, to study the influence of surface modifications on the protein?RNA affinity. The threonine residue in the universally conserved triad Thr?Met?Gly significant for RNA recognition and binding was substituted by phenylalanine, valine and alanine, respectively. The crystal structure of the mutant Thr217Val of the isolated domain I of L1 from Thermus thermophilus (TthL1) was determined. This structure and that of two other mutants, which had been determined earlier, were analysed and compared with the structure of the wild type L1 proteins. The influence of structural changes in the mutant L1 proteins on their affinity for the specific 23S rRNA fragment was tested by kinetic experiments using surface plasmon resonance (SPR) biosensor analysis. Association rate constants undergo minor changes, whereas dissociation rate constants displayed significantly higher values in comparison with that for the wild type protein. The analysed L1 mutants recognize the specific RNA target site, but the mutant L1?23S rRNA complexes are less stable compared to the wild type complexes. Copyright © 2010 John Wiley & Sons, Ltd.     

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 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.     

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.     

The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29.

When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.     

Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5''-end region of the L11 operon.

The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E. coli. Three different sequences were observed in E. coli MRE 600. All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E. coli K12. Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon. This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al. (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1.     

Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl radical probing.

Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.     

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.     

Structure of a protein L23-RNA complex located at the A-site domain of the ribosomal peptidyl transferase centre

Protein L23 from the ribosome of Escherichia coli is the primary ribosomal product cross-linked to affinity-labelled puromycin; it lies, therefore, within the A-site domain of the peptidyl transferase centre on the 50 S subunit. We have characterized this functional domain by isolating and sequencing the RNA binding site of protein L23; it consists of two main fragments of 25 and 105 nucleotides that strongly interact and are separated by 172 nucleotides in the primary sequence. The higher-order structure of the RNA moiety was probed by chemical reagents, and by single-strand and double-strand-specific ribonucleases; a secondary structural model and a tertiary structural interaction are proposed on the basis of these data that are compatible with phylogenetic sequence comparisons.Several nucleotides exhibited altered chemical reactivity, both lower and higher, in the presence of protein L23, thereby implicating a large proportion of the RNA structure in the protein binding. The sites were located mainly at the extremities of the helices and at nucleotides that were putatively bulged out from the helices.The RNA moiety and an adjacent excised fragment contain several highly conserved sequences and a modified adenosine. Such sequences constitute important functional domains of the RNA and may contribute to the putative role of this RNA region in the peptidyl transferase centre.     

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.     

A Novel Association between Two Trypanosome-Specific Factors and the Conserved L5-5S rRNA Complex

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis.     

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.     

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.     

Localization of L11 on the Escherichia coli ribosome by singlet-singlet energy transfer

Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components. The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer. The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm. Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12.