V.A. Erdmann

Structure and Functios of 5 S and 5.8 S RNA

Publisher Summary This chapter reviews that 5S RNA is a small ribonucleic acid associated with the large ribosomal subunits of eukaryotic (60 S) and prokaryotic (5O S) ribosomes. It was soon determined that its sequence did not contain modified nucleotides, such as found in tRNAs. The discovery of 5S RNA was followed by investigations concerning its sequence and physical properties, to obtain information concerning its structure and function. It reviews that the primary structures of 5S RNA cannot be folded in a single pattern, such as the cloverleaf structure of tRNA. After a method for the total reconstitution of bacterial 50S, ribosomal subunits were developed; it became possible to add a new approach to the research concerning the structure and function of 5S RNA. The chapter discusses that the information on eukaryotic 5S and especially 5.8S RNA is scarce and calls for more experimentation. For the near future, detailed investigations concerning the structure of 5S RNA protein complexes seem most promising, as they represent one of the best systems in which to study RNA–protein interactions. Certainly, attempts should be made to crystallize these complexes to obtain the intimate information required for knowing the forces that govern nucleic acid–protein interaction.

The involvement of 5S RNA in the binding of tRNA to ribosomes

Abstract The tRNA fragment TpψpCpGp was found to bind to 5S RNA. This binding is ten times increased when a specific 5S RNA-protein complex is used. The ability of TpψpCpGp to bind to the complex could be abolished by selective chemical modification of two adenines in 5S RNA. Such 5S RNA, when incorporated into 50S ribosomal subunits, yielded particles with greatly reduced biological activities. From the results presented we conclude that 5S RNA is most likely part of a site with which the TψC-loop of tRNA interacts on the ribosome.

Isolation and characterization of 5S RNA-protein complexes from Bacillus stearothermophilus and Escherichia coli ribosomes

SummaryA native B. stearothermophilus 5S RNA-protein complex was isolated. Homologous and hybrid 5S RNA-protein complexes could be reconstituted from B. stearothermophilus and E. coli 5S RNA and ribosomal proteins. The major proteins involved in these complexes are for the B. stearothermophilus system B-L5 and B-L22 and for the E. coli system E-L18 and E-L25. Furthermore, a two-dimensional electrophoresis pattern of B. stearothermophilus 50S proteins is presented.

Active sites in Escherichia coli ribosomes

The model of active sites in E. coli ribosome illustrated in the figure is based on the presently available experimental results. It is far from being complete and should not be overinterpreted as an accurate topographical model. More data on the functional role of ribosomal components and on the topography of the subunits can be expected in the near future and will add to the knowledge on the active sites in ribosomes.

Activities of B. stearothermophilus 50 S ribosomes reconstituted with prokaryotic and eukaryotic 5 S RNA.

Reconstitution experiments have clearly demonstrated the importance of 5 S RNA as a structural component in the 50 S ribosomal subunit [ 1,2]. 50 S ribosomes reconstituted without 5 S RNA are void of several proteins and show greatly reduced biological activities. From these experimental findings it became clear that for studying the biological function of 5 S RNA, modification of the RNA followed by reconstitution and protein synthesizing assays, would be helpful. Two principle types of modifications were at our disposal: i) chemical modification and ii) natural modification as has been undergone in different proand eukaryotic organisms during evolution. Using chemical modification it has been shown that the 3’ terminal base in 5 S RNA does not play an essential role in the structure and function of the 50 S subunit [3,4]. More recently, we could show that selective chemical modification of two adenines per 5 S RNA molecule has significant effects on its function, but not on its structure (Erdmann, Sprinzl and Pongs, in prep aration). The other type of 5 S RNA modification mentioned above, i.e., the natural one, will be analysed here. Earlier experiments have shown that it is possible to use 16 S RNA of different bacterial strains and E. coli 30 S proteins to reconstitute active hybrid 30 S subunits [5]. Similar experiments were carried out with 23 S RNA from Staphylococcus aureus and proteins

Binding oligonucleotides to Escherichia coli and Bacillus stearothermophilus 5 S RNA

Abstract Binding complementary tri- and tetranucleotides to Escherichia coli A19 and Bacillus stearothermophilus 799 5 S RNAs permitted identification of single-stranded regions in these RNAs. Sequences around positions 10, 30, 60, 70, 85 and 95 are in a single-stranded conformation in both 5 S RNAs. It is concluded that the overall structure of bacterial 5 S RNA has been conserved during evolution. Two types of structural conservation have been observed at specific sites of the 5 S RNA: firstly, nucleotide sequence and single strandedness and secondly, single strandedness only. The oligonucleotide binding data for E. coli 5 S RNA are in general agreement with a previous study (Lewis and Doty, 1970) and do not support fully any proposed structural model.

Structure and function of 5S RNA: The role of the 3′ terminus in 5S RNA function

Summary5S RNA from B. stearothermophilus and E. coli was reacted with NaIO4 and aniline to remove their 3′ terminal nucleoside. These modified 5S RNA molecules were then incorporated in B. stearothermophilus 50 S ribosomal subunits and tested for biological activities. 50 S ribosomes containing the modified 5S RNAs exhibited full activity and we therefore conclude, that the 3′ terminus of 5S RNA does not play an active role in protein synthesis.

Irreversible binding of chloramphenicol analogues to E. coli ribosomes.

One of the most obvious dif~culties ln assigning specific functions to individual ribosomal proteins is the great complexity of the ribosom~ particle. Consequently, many different approaches have been tried towards a detailed understand~g of structure-function relationship of the ribosome (for review see [l] ). A promising approach for testing the function of individual ribosomal proteins is the technique of affinity labeling as has been very recently demonstrated by the covalent attachment of peptidyl-tEWA analogues to E. coli ribosomes [2,3]. This report presents the first results of studies which probe chemically the chloramphenicol binding region of E. coli ribosomes by reacting ribosomes with chloramphenicol analogues?. Chloramphenicol has been known for many years as a strong i~ibitor of protein biosynthesis [4], which probably acts at or near the peptidyl-transferase center of the ribosome [.%I Cloramphenicol was modified such that it did not lose its antibiotic specificity. The chemically reactive group introduced into the antibiotic was expected to react preferentially with a properly oriented ammo acid functional group in the binding region of the ribosome. The specificity of the labelling reaction by the chloramphenicol analogues monobromamphenico1 and monoiodoamphenicol was tested i) by compar-

Affinity labeling of E. coli ribosomes with a streptomycin-analogue

Streptomycin inhibits protein synthesis in sensitive bacteria [l]. It has been shown that the inhibitory action of this antibiotic is due to a distortion of the ribosomal-tRNA binding site [2]. Binding of aminoacyl-tRNA and of polypeptidyl-tRNA is impaired, which is followed by a subsequent polysome breakdown [2-4]. Genetic data [5] as well as partial reconstitution experiments [6,7] have shown in one way or the other that several ribosomal proteins are involved in streptomycin binding. Protein S 12 is the ribosomal component which confers resistance against or dependence on streptomycin. Phenotypic reversion from streptomycin dependence to independence can be caused by an altered S 4 or S 5 protein [8]. As suggested by reconstitution experiments, proteins S 3 and S 5 are directly involved in streptomycin binding. Furthermore, the S 5 dependent binding is stimulated by protein S 9 and s 14 [7]. This report presents the first results of studies which probe chemically the streptomycin binding site of E. coli ribosomes by reacting ribosomes with a streptomycin-affinity label.This label was constructed in such a way, that it did not lose its antibiotic specificity, but contained a chemically reactive group, which was expected to react preferentially with a properly oriented amino acid side chain at or near the streptomycin binding region of the ribosome.

The structure of the CCA end of tRNA, aminoacyl-tRNA and aminoacyl-tRNA in the ternary complex.

The 3’.terminal CCA end of tRNA is an essential part of the molecule for its biological function since it participates directly in the following reactions during protein biosynthesis: aminoacylation of tRNA, interaction of aminoacyl tRNA with elongation factor Tu (EF-Tu), binding of aminoacyl-tRNA to the ribosomal A-site, binding of peptidyl-tRNA to the ribosomal P-site and during the stringent response where uncharged tRNA is bound to the ribosomal A-site [ 1 ]. In order to characterize the structure of the 3’-end of tRNA during some of the above-mentioned functional states we have analyzed the accessibility of this part of the molecule by complementary oligonucleotide binding. These results were obtained from using 6 different tRNAs. The analysis included uncharged tRNAs, charged tRNAs and aminoacylated tRNAs involved in the ternary cbmplex with elongation factor Tu and GTP. The results show that the 3’-terminal adenine becomes least accessible towards oligonucleotide interaction after aminoacylation. Addition of EF-Tu re-exposes this adenine and at the same time reduces the accessibility of the fourth base from the 3’.end for oligonucleotide interaction.