A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.     

Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

Isolation of ribosomal protein-RNA complexes by nitrocellulose membrane filtration: equilibrium binding studies.

E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions, making it applicable to weak as well as strong protein-RNA associations. We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 X 10(7)M-1.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.     

Evidence for a precursor-product relationship in the biosynthesis of ribosomal protein S20.

The kinetics of labeling ribosomal protein S20 of Escherichia coli strains H882 and H882 groE44 have been examined using partial reconstitution as a means of binding this and some other 30S subunit proteins selectively to 16S RNA from crude extracts prepared by acetic acid extraction of pulse-labeled whole cells. The rate of labeling of S20 during short pulses at 44 degrees C is less than 20% of that observed at 28 degrees C. S20 can be recovered from the cells labeled at the higher temperature if they are chased at 28 degrees C, but not at 44 degrees C, in the presence of excess sulfate prior to their extraction. These observations suggest that S20 is derived from a precursor whose processing is blocked at 44 degrees C. Among the proteins extracted from cells labeled at 44 degrees C capable of binding to 16S RNA is a novel polypeptide, p2, which is not normally present on the 30S subunit. The kinetics of its appearance at 44 degrees C, and its chasing at 28 degrees C, suggest a precursor-product relationship with S20. p2 contains a tryptic peptide with the chromatographic properties of the peptide Ser-Met-Met-Arg at position 25-28 in S20. A second methionine-containing peptide at positions 49-59 of S20 is missing from p2. In addition, the apparent molecular weight of p2 (8600) is less than that of S20 (9500). p2 may represent the product of degradation of a precursor to S20, yet retains the ability to bind to 16S RNA. It is much less likely that p2 is a bona fide precursor which is converted into S20 by fusion to some other polypeptide.     

Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action.     

Apparent association constants for E. coli ribosomal proteins S4, S7, S8, S15, S17 and S20 binding to 16S RNA.

We have previously reported the development of a technique utilizing nitrocellulose filters, which rapidly separates ribosomal protein-ribosomal RNA complexes from unbound protein. We have used this technique to obtain binding data for the association of proteins S4, S7, S8, S15, S17, and S20 with 16S RNA. With the exception of protein S17, the association behavior for each of these proteins exhibits a single binding site with a unique binding constant. The apparent association constants have been calculated and have been found to have a range from 1.6 x 10(6) M-1 for protein S7 to 7.1 x 10(7) M-1 for protein S17. The Scatchard plot for the protein S17 binding data is biphasic, suggesting that within the RNA population two different binding sites exist, each with a different apparent association constant.     

Significant changes in 16 S RNA conformation accompanying assembly of the 30 S ribosome in vitro

Our previous studies have shown that 16 S RNA can assume two different conformational forms as detected by agarose gel electrophoresis, and that these two forms vary in their ability to bind individual 30 S ribosomal proteins specifically. In this paper we show that the faster electrophoretic form can be converted to the slower electrophoretic form by the binding of either protein S4, S8, S7 or S15. The slower form can then be transformed into a fast form by heat-activating the reconstitution intermediate (RI) particle, which has been constructed under reconstitution conditions at 0 °C, to RI¹. We demonstrate that the transformation of the 16 S RNA conformation by binding of protein S7 permits the subsequent binding of protein S9 following deproteination. We propose that many of the classical assembly-dependent relationships are due to induced changes in the 16 S RNA conformation.     

The Genesis of Ribosome Structure: How a Protein Generates RNA Structure in Real Time

Ribosomal subunit assembly is initiated by the binding of several primary binding proteins. Results from chemical modification studies show that 16S ribosomal RNA undergoes striking structural rearrangements when protein S17 is bound. For the first time, we are able to distinguish and order these structural rearrangements by using time-dependent chemical probing. Initially, protein S17 binds to a portion of helix 11, inducing a kink-turn in that helix that bends helix 7 toward the S17-helix 11 complex in a hairpin-like manner, allowing helix 7 to bind to protein S17. This structural change is rapidly stabilized by interactions at the distal and proximal ends of both RNA helices. Identifying the dynamic nature of interactions between RNA and proteins is not only essential in unraveling ribosome assembly, but also has more general application to all protein-RNA interactions.     

RNA--protein interactions in the ribosome. III. Conformation and stability of ribosomal protein binding sites in the 16 S RNA

Following dialysis against distilled water, the 16 S ribosomal RNA of Escherichia coli is unable to interact with 30 S subunit protein S4 at 0 °C. The dialysed RNA recovered this capacity, however, when heated at 40 °C in the presence of 0.02m-MgCl₂ prior to addition of the protein. Furthermore, its sensitivity to ribo-nuclease markedly declined and its sedimentation rate increased as a consequence of this treatment. Although no concomitant changes in secondary structure were detected by absorbance and fluorescence techniques, the rearrangement of a small number of base-pairs was not excluded. Kinetic measurements revealed that binding site reactivation satisfies the first-order rate law and that the process is highly temperature-dependent, exhibiting an Arrhenius activation energy of 40,800 cal/mol. Together, these data suggest that dialysed RNA undergoes a unimolecular conformational transition upon pre-incubation in Mg²⁺-containing buffers and that this transition leads to renaturation of the binding site for protein S4.Similar results were obtained for several other proteins of the 30 S subunit. In particular, S7, S16/S17 and S20 all failed to interact efficiently with dialysed 16 S RNA at 0 °C. These proteins bound normally to the RNA, however, after it had been incubated at 40 °C in the presence of Mg²⁺ ions. By contrast, prior dialysis of the 16 S RNA did not affect its ability to associate with S8 and S15 at 0 °C. These two proteins interacted equally well with dialysed and pre-incubated 16 S RNA, indicating that their binding sites are not susceptible to the reversible alterations in conformation which influence the attachment of the other RNA-binding proteins to the nucleic acid molecule. The effects of dialysis and pre-incubation on the interaction of 16 S RNA with an unfractionated mixture of 30 S subunit proteins were also investigated. The dialysed RNA bound only S6, S8, S15 and S18 at 0 °C whereas, after heating at. high Mg²⁺ concentrations, the RNA associated with S4, S7, S9, S13, S16/S17, S19 and S20 as well. These results leave little doubt that the protein-binding capacities of the 16 S RNA are intimately related to its three-dimensional configuration, although individual binding sites appear to differ significantly in their stability to small changes in structure.     

Chemical and functional characterization of an altered form of ribosomal protein S4 derived from a strain of E. coli defective in auto-regulation of the alpha operon.

We have isolated a mutant form of Escherichia coli ribosomal protein S4. This mutant is temperature sensitive and apparently fails to autogenously regulate the gene products of the alpha operon, which consists of the genes for proteins S13, S11, S4, L17, and the alpha subunit of RNA polymerase (1). We have shown that this mutation results in the production of an S4 protein with a molecular weight approximately 4,000 daltons less than the wild-type protein. Our chemical analyses demonstrate that the mutant protein is missing its C-terminal section consisting of residues 170-203. However, our studies to determine the capacity of this mutant protein to bind 16S RNA show that this protein is unimpaired in RNA binding function. This observation suggests that the functional domain of protein S4 responsible for translational regulation of the S4 gene products requires more of the protein than the 16S RNA binding domain.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Chemical inactivation of Escherichia coli 30-S ribosomes by iodination. Identification of proteins involved in tRNA binding.

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non-enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles shown normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect.     

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.     

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.     

The structure of the RNA binding site of ribosomal proteins S8 and S15.

Proteins S8 and S15 from the 30 S ribosomal subunit of Escherichia coli were bound to 16 S RNA and digested with ribonuclease A. A ribonucleoprotein complex was isolated which contained the two proteins and three noncontiguous RNA subfragments totaling 93 nucleotides, that could be unambiguously located in the 16 S RNA sequence. We present a secondary structural model for the RNA moiety of the binding site complex, in which the two smaller fragments are extensively base-paired, respectively, to the two halves of the large fragment, to form two disconnected duplexes. Each of the two duplexes is interrupted by a small internal loop. This model is supported by (i) minimum energy considerations, (ii) sites of cleavage by ribonuclease A, and (iii) modification by the single strand-specific reagent kethoxal. The effect of protein binding on the topography of the complex is reflected in the kethoxal reactivity of the RNA moiety. In the absence of the proteins, 5 guanines are modified; 4 of these, at positions 663, 732, 733, and 741, are strongly protected from kethoxal when protein S15 is bound.     

Ribosomal protein-nucleic acid interactions. I. Isolation of a polypeptide fragment from 30S protein S8 which binds to 16S rRNA.

Within the bacterial ribosome a large number of specific protein and rRNA interactions appear to be required for assembly of the particle and its subsequent function in protein synthesis. In this communication it is shown that it is possible to isolate cyanogen bromide digestion products from ribosomal 30S protein S8 which will interact stoichiometrically with 16S rRNA. In addition to this a small binding polypeptide was generated from S8-16S rRNA complexes which were treated with proteinase K. The digestion of the complex yields a "protected" fragment of protein S8 which binds to 16S-rRNA. The isolated fragment will reassociate with 16S rRNA. It is not displaced by other 30S ribosomal proteins and blocks the binding of intact S8 to 16S rRNA. The size the possible structure of the S8 protein binding site are discussed and compared with the binding of cyanogen bromide digestion products which bind to 16S rRNA.     

Evidence that E. coli ribosomal protein S13 has two separable functional domains involved in 16S RNA recognition and protein S19 binding.

We have found that E. coli ribosomal protein S13 recognizes multiple sites on 16S RNA. However, when protein S19 is included with a mixture of proteins S4, S7, S8, S16/S17 and S20, the S13 binds to the complex with measurably greater strength and with a stoichiometry of 1.5 copies per particle. This suggests that the protein may have two functional domains. We have tested this idea by cleaving the protein into two polypeptides. It was found that one of the fragments, composed of amino acid residues 84-117, retained the capacity to bind 16S RNA at multiple sites. Protein S19 had no affect on the strength or stoichiometry of the binding of this fragment. These data suggest that S13 has a C-terminal domain primarily responsible for RNA recognition and possibly that the N-terminal region is important for association with protein S19.     

Binding of Escherichia coli ribosomal proteins to 23S RNA under reconstitution conditions for the 50S subunit.

The RNA binding capacity of 50S proteins from E. coli ribosomes has been tested under improved conditions; purified proteins active in reconstitution assays were used, and the binding was studied under the conditions of the total reconstitution procedure for the 50S subunit. The results are: 1) Interaction of 23S RNA was found with 17 proteins, namely L1, L2, L3, L4, L7/L12, L9, L10, L11, L15, L16, L17, L18, L20, L22, L23, L24 and L29. 2) The proteins L1, L2, L3, L4, L9, L23 and L24 bound to 23S RNA at a level of about one copy per RNA molecule, whereas L20 could bind more than one copy (no saturation was observed at 1.8 copies per 23S RNA), and the other proteins bound 0.2--0.6 copies per RNA. 3) L1, L3, L7/L12 showed a slight binding to 16S RNA, L26 (identical with S20) strong binding to 16S RNA. 4) The binding of L2, L7/L12, L10, L11, L15, L16 and L18 was preparation sensitive, i.e. the binding ability changed notably from preparation to preparation. 5) All proteins bound equally well to 23S RNA in presence of 4 and 20 mM Mg2+, respectively, except L2, L3, L4, L7/L12, L9, L10, L15, L16 and L18, which bound less strongly at 20 mM than at 4 mM Mg2+.     

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.