Roger A. Garrett

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

SummaryThe identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg++ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fabs) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgGs specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.

Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 S RNA and specific 50 S subunit proteins

Abstract Pancreatic RNase partial digests of 32 P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed.

Ribosomal proteins. XXVI. The number of specific protein binding sites on 16 s and 23 s RNA of Escherichia coli

Abstract The number of specific binding sites for proteins on 16 s and 23 s ribosomal RNA of Escherichia coli was investigated. Immunochemical and acrylamide gel methods were used for the detection of binding. On the 16 s rRNA, five sites, for proteins S4, S7, S8, S15 and S20, were found. On the 23 s rRNA, eight sites were detected, for proteins L2, L6, L16, L17, L19, L20, L23 and L24. Moreover, one 30 s protein, namely S11, bound specifically to 23 s rRNA.

Binding of 50 S ribosomal subunit proteins to 23 S RNA of Escherichia coli

Each of the 50 S ribosomal subunit proteins of Escherichia coli was tested independently in two laboratories for its ability to bind specifically to 23 S RNA. Four new RNA-binding proteins, L1, L3, L4 and L13 were identified in this way. Consistent with earlier work, proteins L2, L6, L16, L20, L23 and L24 were found to interact directly and independently with 23 S RNA as well. No binding of L17 was detected, however, contrary to previous reports, and the results for L19 were variable. The molar ratio of protein and RNA in each complex was measured at saturation. Significant differences in binding stoichiometry were noted among the various proteins. In addition, saturation levels were found to be influenced by the state of both the RNA and the proteins.

The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding

SummaryThe accessibility of each of the proteins on the E. coli 30S ribosomal subunit was established by investigating whether or not immunoglobulins (IgGs) and their monovalent papain fragments (Fabs), specific for each of the 21 single ribosomal proteins, bind to the 30S subunit. The interpretation of the results of five different experimental approaches, namely Ouchterlony double diffusion and immunological “sandwich” methods, sucrose gradient and analytical ultracentrifugation, and functional inhibition tests, indicate that all 21 proteins of the 30S subunit have determinants available for antibody binding. There were quantitative differences between the degree of accessibility of the different ribosomal proteins. An attempt was made to correlate the results with the protein stoichiometric data of the small subunit proteins.

Structure of bacterial ribosomes.

Publisher Summary This chapter discusses the physical and chemical properties of all the individual ribosomal proteins present in Escherichia coli bacteria. It also describes the mechanism of protein synthesis. Escherichia coli ribosomes contain 55 proteins in addition to three RNA molecules. The separation and characterization of the individual proteins is important for assessing the complexity of the ribosome and for the detailed structural and functional investigations of the ribosomes. Experimentally, the isolation of individual proteins proved to be very difficult; first, because many of the proteins have similar chemical and physical properties and second, because the mixture of proteins was relatively insoluble and would dissolve readily only under extreme pH conditions, where there is the possibility of the chemical modification of the proteins, or at high urea concentrations, where the proteins may be denatured. The molecular weights of E. coli ribosomal proteins are determined by equilibrium sedimentation and by poly acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Structure of 5 S ribosomal RNA from Escherichia coli: Identification of kethoxal-reactive sites in the A and B conformations☆

Abstract The topographies of the A and B conformers of free 5 S RNA have been examined using kethoxal as a probe of single-stranded, accessible guanine residues. Each of the kethoxal-reactive guanines has been identified using diagonal electrophoresis, and the relative rate of modification at each site has been studied. Free 5 S RNA in the A form has several reactive guanines in addition to G13 and G41, which are the only two available for reaction in the intact 50 S ribosomal subunit (Noller & Herr, 1974). The relative reactivities of these sites are G41 ⪢ G13 > G69 > G24 > G86 > G107 > G16, G23, G44. Modification at G23 and G44 reaches maximum values of only about 0.05 mol per mol 5 S RNA, suggesting that these residues are unreactive in the major conformer of the A form population. These results are compatible with a secondary structure model based on phylogenetic sequence conservation (Fox & Woese, 1975), but imply that 12 of the 18 unpaired guanines in this model are involved in further molecular interactions. The modification pattern of the B conformer demands a different base-pairing arrangement and shows that the B form contains less structure than the A form. The relative reactivities in the B form are G13 > G102 > G16 > G24, G44 > G61, G100 > G23, G51, G107 > G54, G56. Several sites show plateaux at submolar modification levels, indicating the existence of some conformational heterogeneity in preparations of the B form of 5 S RNA. Heat-denatured 5 S RNA appears to contain a mixture of conformers including the A and B form. These results place limitations on certain structural and functional models for 5 S RNA. For example, G44, which has often been implicated in base-pairing with tRNA, is accessible in the B form but not in the A form. Yet the B form does not bind the 5 S RNA-specific ribosomal proteins, nor is there evidence for its existence in the ribosome.

Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA.

Abstract The topography of Escherichia coli 5S RNA has been examined in the presence of ribosomal proteins L5, L18 and L25 and their different combinations, by comparing the kethoxal modification characteristics of the various RNA-protein complexes with those of the free A-conformer of 5S RNA (Noller & Garrett, 1979, accompanying paper). Two of the four most reactive guanines, G13 and G41, are unaffected by the protein, in accord with the finding that these are the only two guanines that are accessible in the 50S subunit (Noller & Herr, 1974). The other two very reactive guanines, G24 and G69, are strongly protected by protein L18, either in the presence or absence of proteins L5 and L25. Protein binding studies with kethoxal-modified 5S RNA provide evidence that one or both of these two guanines are directly involved in the protein-RNA interactions, and this conclusion is supported by the occurrence of guanines in these two positions in all the other sequenced prokaryotic 5S RNAs. The group of less reactive guanines, G16, G23, G44, G86 and G107, are protected to some extent by each of the proteins L5, L18 and L25; the strongest effect is with L18. We suggest that this is attributable to a small increase in the conformational homogeneity of the 5S RNA and that L18, in particular, induces some tightening of the RNA structure. Only one guanine, G69, is rendered more accessible by the proteins. This effect is produced by protein L25, which is known to cause some destructuring of the 5S RNA (Bear et al., 1977). There was no other evidence for any destructuring of the 5S RNA. In particular, the sequence 72 to 83, which is complementary to a sequence in 23S RNA (Herr & Noller, 1975), is not modified. However, in contrast to an earlier report (Erdmann et al., 1973), the conserved sequence G44-A-A-C, which has been implicated in tRNA binding, was not rendered more accessible by the proteins.

Small-angle X-ray scattering study of the 16 S RNA binding protein S4 fromEscherichia coli ribosomes

It is indicated from recent small-angle X-ray scattering studies [l-3] that the L18, L2.5 and L7/Ll2 ribosomal proteins are highly elongated. Similar conclusions have recently been drawn for many other ribosomal proteins using other methods. For instance, electron microscope studies using antibody markers [4,5] show that proteins S4, SS, Sll, S12, and perhaps also S2, S7, Sl5 and S18 are elongated; neutron scattering data indicate protein S2 to be elongated [6]. However, very little is known regarding how these highly elongated proteins are packed within the ribosomal subunits; previous ribosome models are based almost entirely on spherical protein models [7]. An idea of how the various riposomal components might be packed together seemi to be emerging from small-angle X-ray scattering studies of protein-protein complexes and protein-RNA complexes [8,9] ; for instance, the L7/L12-LlO complex [8] appears to have a conformation similar to an elongated, flattened disc, and the S4 binding site on 16 S RNA [lo] appears to be a flattened, oblate ellipsoid. This report deals with a separate study of the protein S4 from the 30 S subunit. The main results indicate that protein S4, by itself, has a conformation very similar to a flat, elongated ellipsoid with the semiaxes a = 90, b = 25, and c = 4 A. The protein S4 was prepared via two different

The identification of new RNA-binding proteins in the Escherichia coli ribosome.

Identifying those proteins that bind independently to ribosomal RNAs of the E’, cofi ribosome has proved an important step in the structural characterization of the ribosome. The RNA binding site can subsequently be localised within the RNA sequence and, in some instances, it has been possible to isolate a complex of the protein and its RNA binding site which can then be subjected to detailed structural studies (reviewed in [ 1,2]). There is general agreement that proteins S4, S7, SS, SlS and S20 specifically bind to phenol-extracted 16 S RNA and that proteins Ll, L2, L3, L4, L6, L16, L20, L23 and L24 bind to the 23 S RNA [ 1,3]. Conflicting results have appeared on the binding of S13 and S17 to 16 S RNA and L13 and L19 to 23 S RNA [ 1,3]. Recently, Hochkeppel et al. [4] demonstrated that a further seven proteins from the 30 S subunit, namely S3, SS, S9, Sll, S12, S13 and S18, could bind to 16 S RNA that was prepared by a low pH extraction procedure. The binding of these proteins was attributed to the formation of a metastable and more open RNA conformation. In the present study we have tested 34 ribosomal proteins for specific RNA-binding which were fractionated by very gentle procedures that differed from previous preparation methods in that they avoided any protein denaturing conditions [S]. RNAs, 16 S and 23 S, were prepared by the standard phenoldodecylsulphate method. The binding results demonstrate that in addition to the well characterized RNA-binding proteins, ribosomal proteins S2, SS, S13 and S19 bind to the 16 S RNA and proteins Lll and Ll S bind to 23 S RNA. These results establish that the conformations of the proteins are also very important for attaining stable protein-RNA interactions. Preliminary evidence for the binding of proteinprotein complexes to the ribosomal RNAs is also presented.