Gary R. Craven

Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA.

We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.

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

Abstract 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.

Purification and characterization of 50S ribosomal proteins of Escherichia coli

SummaryTwenty-seven proteins of the 50S ribosomal subunit from E. coli have been purified by a combination of differential solubility in ammonium sulfate, ion-exchange chromatography, and molecular-sieve chromatography. The amino acid compositions, tryptic peptides and molecular weights of these proteins have been analyzed. Each protein is unique with respect to amino acid sequence and, according to chemical criteria, reasonably pure. The sum of the molecular weights of the twenty-seven proteins is 495000. This means that the 50S subunit could accommodate one copy of each protein.

The function of the N-terminal region of ribosomal protein S4

Single ribosomal protein-16 S RNA complexes were examined for their sensitivity to trypsin digestion under conditions which maintain specific protein-RNA interactions. Proteins S7 and S13 are completely digested and removed from the complex while proteins S8, S15, S17 and S20 are protected from trypsin attack by the 16 S RNA. Protein S4 is partially protected yielding a fragment of a molecular weight of about 17,700. These results are in agreement with similar experiments done with different digestion conditions by Schulte et al. (1975). We have determined that the portion of protein S4 removed by the trypsin is the N-terminus up to residues 43 to 46. This conclusion is based on the following experimental results: (1) the tryptophan residue at position 167 remains a part of the S4 fragment after trypsin digestion; (2) the cysteine residue at position 31 is removed by trypsin digestion; (3) tryptide fingerprints indicate that the tyrosine at position 50 is not lost by trypsin digestion; (4) the loss of 4800 daltons is equivalent to approximately 44 amino acid residues. The role the N-terminus of protein S4 plays in ribosome assembly and function was investigated. Protein S4 fragment was mixed with 20 purified 30 S ribosomal proteins excluding protein S4 and reconstituted with 16 S RNA. An incomplete particle was produced. The proteins completely missing are S2, S10, S18 or S19 and S21. We postulate that these proteins interact either directly or indirectly with the N-terminal region of S4. Correlating this hypothesis with the electron microscopic results of Tischendorf et al. (1975), we also suggest that the N-terminus of protein S4 resides in the “head” region of the electron microscope model.

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.

Studies on the role of amino acid residues 31 through 46 of ribosomal protein S4 in the mechanism of 30 S ribosome assembly.

Abstract We have described previously the isolation of a large fragment of 30 S ribosomal protein S4 (Changchien & Craven, 1976). This S4-fragment is produced by the digestion of the S4–16S RNA complex with trypsin and it retains a full capacity to associate specifically with 16S RNA. It was also demonstrated that the S4-fragment has approximately 46 amino acid residues missing from the N-terminus and an intact C-terminus (also shown by Newberry et al. , 1977). Preliminary experiments with this S4-fragment indicated that it could not fully replace the intact protein S4 in the process of 30 S ribosome assembly in vitro . We have also recently reported (Changchien et al. , 1978) the preparation of a new fragment of protein S4 which has only 30 amino acid residues cleaved from the N-terminus. This was achieved by the use of the reagent 2-nitro-5-thiocyanobenzoic acid which selectively modifies the cysteine residue at position 31 followed by a cleavage of the adjacent peptide bond. We have now fully characterized the capacity of these two fragments, S4-fragment (47–203) and S4-fragment(31–203), to participate in the 30 S ribosome assembly process in vitro . Using 2-dimensional polyacrylamide gel electrophoresis, we find that when S4-fragment(47–203) is a component of the in vitro assembly reaction, proteins S1, S2, S10, S18 and S21 fail to become incorporated into the final particle. In contrast, S4-fragment(31–203) appears to participate in the reconstitution reaction without impairment allowing the complete incorporation of all 20 proteins of the 30 S subunit. The resultant particle, containing the S4-fragment (31–203), is fully active in the binding of poly(U), but is completely inactive for non-enzymatic poly(U)-directed binding of Phe-tRNA (Changchien et al. , 1978). These results suggest that residues 1 through 30 of protein S4 are not involved in the assembly of the 30 S ribosome, but are required for the proper construction of the tRNA binding site. In addition residues 31 through 46 must be somehow critically important for the assembly of proteins S1, S2, S10, S18 and S21. We present evidence to show that the absence of residues 31 through 46 of protein S4 prevents a conformational change in the structure of 16 S RNA which normally accompanies the RI to RI transition and that this results in the inability of these proteins to participate in the assembly process.

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 RI∗. 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.

Identification of several proteins involved in the messenger RNA binding site of the 30 S ribosome by inactivation with 2-methoxy-5-nitrotropone.

Abstract Chemical modification of unwashed 30 S ribosomal subunits with 2-methoxy-5-nitrotropone causes a rapid loss of their capacity to bind bacteriophage Qβ RNA. Reconstitution experiments show that ribosomal protein is the functionally inactivated species. When purified unmodified ribosomal proteins were included in a mixture of 16 S ribosomal RNA and total protein derived from 2-methoxy-5-nitrotropone-treated subunits, four proteins (S1, S12, S13 and S21) were found to promote the reconstitution of particles capable of binding natural messenger RNA.

Proximity relationships among the 30 S ribosomal proteins during assembly in vitro

Abstract Previous studies (Craven et al. , 1974) demonstrated that the capacity of ribosomal proteins to be chemically modified by iodine is extensively reduced when they are members of an intact ribosome. We have attempted to exploit this observation by analyzing in detail the alterations in the iodine accessibility of the individual 30 S ribosomal proteins. We have prepared a total of 38 different complexes between 16 S RNA and mixtures of individual purified 30 S ribosomal proteins. Eighteen of the 21 30 S proteins were used in the formation of these complexes. Comparison of the iodination patterns obtained for the various proteins derived from different complexes has revealed that sometimes a specific protein can selectively alter the chemical reactivity of another protein in the complex. We have found 30 different examples of protein pairs in which one protein effectively protects another protein from chemical iodination.

Chemical inactivation of Escherichia coli 30 s ribosomes with maleic anhydride: Identification of the proteins involved in polyuridylic acid binding☆

Abstract Modification of 30 S ribosomal subunits by the protein-modifying reagent maleic anhydride was found to inactivate the particles for polyuridylic acid binding. Reconstitution of 30 S ribosomes using 16 S RNA, maleylated total 30 S protein, and purified, unmodified proteins demonstrated that S4, S11, S12, S13 and S18 are involved in poly(U) binding. Modified 30 S subunits contain all the ribosomal proteins and show normal sedimentation characteristics, indicating that the inactivation is not simply due to the gross alteration of the particles. Correlation of these results with those of other workers is discussed.