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.     

Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.     

Binding sites of rat liver 5S RNA to ribosomal protein L5

The ribonucleoprotein complex consisting of 5S RNA and the protein L5 was prepared from the large subunit of rat liver ribosomes. The RNA in the complex was digested in situ with RNase A or RNase T1. The RNase-resistant RNA fragments bound to the protein were recovered and purified by 2D-PAGE, and their nucleotide sequences were determined in order to elucidate the binding sites of the RNA to the protein. The results showed that the fragments had arisen from the 5'-end region (residues 1-21), from the second hairpin loop (residues 77-102) and from the 3'-end region (residues 106-120). Harsher digestion trimmed these fragments to shorter fragments. It was concluded that the minimal interactive sequences of 5S RNA to the protein L5 were residues 13-21, residues 85-102, and residues 106-114. A part of the first hairpin loop, residues 41-52, was also suspected to interact with the protein. These protein-binding sites of rat liver 5S RNA were compared with those of Escherichia coli, Halobacterium cutirubrum and yeast, and their probable conservation from eubacteria to eukaryotes is discussed.     

A trypsin-resistant fragment from complexes of ribosomal protein S4 with 16-S RNA of Escherichia coli and from the uncomplexed protein.

A fragment of ribosomal protein S4 was prepared by limited trypsin degestion of a specific complex between protein S4 and 16-S RNA. It was characterised for amino acid sequence and the N-terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg-43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA-binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N-terminal region of the protein is proposed and two probable functions of the excised region are given. Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg-46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein-RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.     

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.     

The characterisation of a fragment of ribosomal protein S4 that is protected against trypsin digestion by 16S ribosomal RNA of Escherichia coli.

After mild trypsin treatment of a complex of ribosomal protein S4 and 16S RNA of Escherichia coli, a large homogeneous fragment of the S4 protein was protected against digestion by its RNA binding site. This fragment was isolated and characterised for molecular weight. It was able to rebind specifically to 16S RNA. Preliminary results indicate that protected protein fragments can also be obtained from other proteins that complex specifically with 23S and 5S RNA.     

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.     

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.     

Studies on the primary structure of the ribosomal 23S RNA of Escherichia coli: II. A characterisation and an alignment of 24 sections spanning the entire molecule and its application to the localisation of specific fragments.

We have employed new methodology to obtain 23S RNA fragments which includes a) the digestion of the RNA within 50S subunits and b) the limited hydrolysis of the 13S and 18S fragments. By comparing all 23S RNA fragments, obtained heretofore, we have characterised and aligned 24 sections of this RNA spanning nearly the entire molecule. These results allow the localisation of any new 23S RNA fragment by comparison of the fingerprint of its T1 ribonuclease digest to the characteristic ones of the different sections. In this way we obtained a more definite localisation of the binding sites of the 50S proteins L1, L5, L9, L18, L20, L23 and L25. We also specified a ribonuclease sensitive region of 23S RNA in native 50S subunits, extending from the 1100th nucleotide from the 5' end to the 1000th nucleotide from the 3' end; this region contains a cluster of 5 modified nucleotides and may be at the subunit interface.     

Structural studies of the contractile tail sheath protein of bacteriophage T4. 2. Structural analyses of the tail sheath protein, Gp18, by limited proteolysis, immunoblotting, and immunoelectron microscopy

The molecular structure of the T4 phage tail sheath protein, gp18, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gp18 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsin- and thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gp18 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thr1-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gp18 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51-73].     

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 N-terminal region of the plasma membrane Ca(2+) pump does not separate from the main catalytic fragments after proteolysis

The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.     

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 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

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.