Li-Ming Changchien

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.

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5 domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3 minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5 domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5 and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.

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.

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.

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.

Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous.

We have estimated the number of sites on each protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinatable sites was determined for the intact 30 S ribosome. The proteins were extracted, separated and the relative distribution of iodine in each protein determined. This distribution of iodine divided into the total sites per ribosome gave an estimate of the number of sites per individual protein. n nSecond, the iodinated proteins were purified and their trypsin digestion products separated. The number of radioactive peptides was taken as a measure of the number of sites on that protein open to the iodination reaction. The number of iodinatable sites for each protein was found to be radically different by the two methods. In almost all cases, the number of unique, radioactively labeled peptides, derived from a given 30 S protein, far exceeded the total incorporation into that protein. We suggest that the best explanation for this unexpected discrepancy is that the 30 S ribosome population we used in these experiments is heterogeneous in its topography. n nIn addition we have compared the topography by the chemical iodination procedure for ribosomes in two different conformations: active and inactive (see Zamir et al., 1971). We have found very little change in the chemical reactivity of the proteins when the ribosomes are in the two different conformations. The most notable changes involve proteins S10, S18S19 and especially S12S13.

[16] Isolation of fragments of ribosomal proteins that recognize rRNA

Publisher Summary This chapter focuses on the isolation of fragments of ribosomal proteins that recognize rRNA. Studies on the interactions between ribosomal proteins (r-proteins) and rRNA, as well as specific roles of individual proteins in further assembly (especially their interactions with other proteins) and the functions of the complete ribosome, have become possible after the development of the in vitro reconstitution systems for 30S and 50S ribosomes. This chapter discusses to develop the chemical and enzymatic cleavage techniques to produce fragments of ribosomal proteins which retain specific binding activities for rRNA. It was experimented with several chemical techniques for the fragmentation of ribosomal proteins and had found four cleavage procedures that produce biochemically functional fragments: (1) chemical cleavage at the peptide bond of cysteine after cyanylation with 2-nitro-5-thiocyanobenzoic acid, (2) cleavage at the tryptophanyl peptide bond with a mixture of dimethyl sulfoxide and hydrogen bromide, (3) cleavage of the Asparaginylglycyl (Asn-Gly) peptide bond with hydroxylamine, and (4) cyanogen bromide cleavage of proteins. This chapter also presents the result that have shown that individual ribosomal proteins can be cleaved to produce smaller polypeptides capable of specific RNA association, and that often further functional activities in assembly and translation are lost.

[17] Isolation of kinetic intermediates in in vitro assembly of the Escherichia coli ribosome using cibacron blue F3GA

Publisher Summary This chapter describes the isolation of kinetic intermediates in in vitro assembly of the Escherichia coli ribosomes using Cibacron blue F3GA. Using a column of Cibacron Blue-bound agarose, it is shown that all E. coli ribosomal proteins, when free in solution, bind to the column even in the presence of the high salt concentrations in the reconstitution buffers used in these experiments. Two-dimensional PAGE analysis of 50S reconstitution intermediates isolated following addition of Cibacron Blue at different times during assembly. For the 0 time experiment, the dye was added to the proteins immediately prior to the addition of the RNA. A dye: protein molar ratio of 50: l was used. A judicious use of Cibacron Blue F3GA can make it a simple and valuable tool in isolating kinetic intermediates in the assembly pathway of ribosomes and other systems where interactions between a nucleic acid and multiple proteins are involved. The isolation and analysis of such interm dimes should further ones understanding of biological assembly mechanisms.

[46] Some methods using protection from chemical and enzymic modification to determine protein-protein relationships in ribosomes

Publisher Summary This chapter discusses methods to determine protein-protein relationships in ribosomes using protection from chemical and enzymic modification. Escherichia coli is used throughout in the chapter as an example to illustrate the methods of chemical and enzymic protection for detecting protein-protein relationships. The 30 S bacterial ribosome can be completely reassembled from a mixture of individually purified 30 S proteins and the component 16 S RNA. The technique to construct many different intermediate protein-RNA complexes makes it possible to compare two separate protein-RNA complexes differing in protein content by only a single protein. The protection approach involves the modification of two such protein-RNA complexes, either with a protein selective reagent 5 or a proteolytic enzyme, and analysis of the modification products to determine whether or not the presence of the extra protein in the second complex protected any of the other component proteins from modification. To illustrate the basic procedure for proteolytic protection, the chapter presents a specific series of experiments.

Chemical Approaches to the Analysis of Ribosome Architecture

INTRODUCTION The present state of knowledge of ribosome structure bears a clear resemblance to the problem of enzyme structures as it developed prior to the impact of X-ray crystallography. During that period investigators utilized virtually any techniques they could imagine to uncover the fine details of protein architecture. One of the most widely applied methods to obtain significant information about protein structure has been the utilization of chemicals capable of selectively derivatizing polypeptide side chains. Over the years a substantial number of these reagents have been studied for their ability to react more or less selectively with amino acid functional groups. These reagents are used to determine which amino acid side groups are involved in the function of the enzyme and also to differentiate between so-called “buried” and “exposed” side chains. These data, combined with primary sequence information, have yielded considerable general knowledge of the three-dimensional structure of numerous proteins. Application of Protein Modifying Reagents to the Problem of Ribosome Structure A number of workers have attempted to extend the use of chemical reagents to determine some general features of ribosome structure (Acharya and Moore 1973; Craven and Gupta 1970; Ginzburg, Miskin and Azmir 1973; Huang and Cantor 1972; Hsiung and Cantor 1973; Kahan and Kaltschmidt 1972; Michalski, Sells and Morrison 1973; Miller and Sypherd 1973; Visentin, Yaguchi and Kaplan 1973). The initial effort with ribosomes has been to search for some gross reflections of protein organization within the ribosome architecture. Thus a fair number of different protein reagents have...