The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit

Physical properties of ribosomal proteins obtained with or without denaturating agents were compared. CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents. CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions. These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation.     

Identification of fifteen neighboring protein pairs in the Escherichia coli 50 S ribosomal subunit crosslinked with 2-iminothiolane.

The 50 S ribosomal subunit of Escherichia coli was allowed to react with 2-iminothiolane under conditions in which amidine-linked sulfhydryl derivatives were formed between lysine ?-amino groups in ribosomal proteins and the heterocyclic thioimidate. Crosslinking between sulfhydryl groups close enough to form intermolecular disulfide bonds was promoted by oxidation of the modified ribosomal subunits. Disulfide-linked dimers were partially purified by extraction of the oxidized subunits with lithium chloride and electrophoresis of the salt-extracted fractions in polyacrylamide/urea gels at pH 5.5. Crosslinked protein dimers were separated by polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Fifteen protein dimers were identified. Many of them involve proteins implicated in functional sites of the 50 S subunit and in ribosome assembly. The crosslinking results show the proximity of many of these proteins at these active centers, and extend the neighborhood by demonstrating the presence of additional proteins.     

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.     

Secondary structures of proteins from the 30S subunit of the Escherichia coli ribosome.

The secondary structures of the proteins S4, S6, S8, S9, S12, S13, S15, S16, S18, S20 and S21 from the subunit of the E. coli ribosome were predicted according to four different methods. From the resultant diagrams indicating regions of helix, turn, extended structure and random coil, average values for the respective secondary structures could be calculated for each protein. Using the known relative distances for residues in the helical, turn and sheet or allowed random conformations, estimates are made of the maximum possible lengths of the proteins in order to correlate these with results obtained from antibody binding studies to the 30S subunit as determined by electron microscopy. The influence of amino acid changes on the predicted secondary structures of proteins from a few selected mutants was studied. The altered residues tend to be structurally conservative or to induce only minimal local changes.     

The crystallization of ribosomal proteins from the 50 S subunit of the Escherichia coli and Bacillus stearothermophilus ribosome

Several individual intact ribosomal proteins purified from bacterial sources under mild conditions have been crystallized. A number of these are suitable candidates for three-dimensional structural studies by x-ray diffraction techniques. Data collection to 3 A resolution for one of these proteins is in progress.     

The 30 S subunit of the Escherichia coli ribosome. Topographical model of its component proteins.

This topographical model of the proteins of the 30 S subunit of the Escherichia coli ribosome was built to be consistent with the 37 published spectroscopic and chemical experiments that indicate proximity and with the two neutron diffraction experiments that indicate S3 and S7 as well as S2 and S5 to be separated by 110 A. The model is quite consistent with the protein arrangement suggested by assembly pathways, the various RNA binding sites, and the streptomycin-associated proteins, This consistency is encouraging since none of these data were considered during the construction of the model. The model differs significantly from those proposed by Traut et at. ((1974) Ribosomes 271-308) and by Tischendorf et al. ((1975) Proc. Natl. Acad. Sei. U.S. 72, 4820-4824).     

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive ³⁵S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 mm-Mg²⁺. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated ³⁵S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.     

Identification of neighboring protein pairs in the Escherichia coli 30 S ribosomal subunit by crosslinking with methyl-4-mercaptobutyrimidate

The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.     

The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by reaction with 2-iminothiolane followed by a mild ultraviolet irradiation treatment. After removal of non-reacted protein and partial nuclease digestion of the cross-linked 16S RNA-protein moiety, a number of individual cross-linked complexes could be isolated and the sites of attachment of the proteins to the RNA determined. Protein S8 was cross-linked to the RNA at three different positions, within oligo-nucleotides encompassing positions 629-633, 651-654, and (tentatively) 593-597 in the 16S sequence. Protein S7 was cross-linked within two oligonucleotides encompassing positions 1238-1240, and 1377-1378. In addition, a site at position 723-724 was observed, cross-linked to protein S19, S20 or S21.     

Interactions between 30s ribosomal proteins and 50s subunits of Escherichia coli.

The interaction between ribosomal proteins of the 30s subunit with intact 50s subunits was investigated. Experiments with mixtures of total 30s proteins indicated that several 30s proteins including protein s4 would form a stable complex with 50s subunits. Further work with pure s4 indicates that this protein binds stoichiometrically to the 50s subunits, probably through protein-nucleic acid interaction. The possible significance of this interaction is discussed.     

Topography of the Escherichia coli 30 S ribosome revealed by the modification of ribosomal proteins

Chemical and enzymatic iodination were used to determine the position of proteins within the 30 S ribosome of Escherichia coli. The relative degree of iodination was determined in intact 30 S ribosomes, particles unfolded with EDTA, ribonuclease digests of ribosomes and extracted proteins. These procedures permitted an evaluation of the influence of protein-RNA and protein-protein interactions, protein conformation and position, on the degree to which protein could be modified by radioactive iodine. From the data we conclude that all 30 S ribosomal proteins are accessible to the external milieu, and that none are buried within the three-dimensional structure of the particle.     

Area of 16S ribonucleic acid at or near the interface between 30S and 50S ribosomes of Escherichia coli.

To determine the region of 16S ribonucleic acid (RNA) at the interface between 30 and 50S ribosomes of Escherichia coli, 30 and 70S ribosomes were treated with T1 ribonuclease (RNase). The accessibility of 16S RNA in the 5' half of the molecule is the same in 30 and 70S ribosomes. The interaction with 50S ribosomes decreases the sensitivity to T1 RNase of an area in the middle of 16S RNA. A large area near the 3' end of 16S RNA is completely protected in 70S ribosomes. The RNA near the 3' end of the molecule and an area of RNA in the middle of the molecule appear to be at the interface between 30 and 50S ribosomes. One site in 16S RNA, 13 to 15 nucleotides from the 3' end, normally inaccessible to T1 RNase in 30S ribosomes, becomes accessible to T1 RNase in 70S ribosomes. This indicates a conformational change at the 3' end of 16S RNA when 30S ribosomes are associated with 50S ribosomes.     

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

Pancreatic RNase partial digests of ³²P-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.     

Physical studies on proteins L3 and L24 from the 50 S subunit of the Escherichia coli ribosome.

Proteins L3 and L24, purified by a nondenaturing method from the 50 S ribosomal subunit of Escherichia coli A19, have been characterized. Both proteins were studied under conditions which resemble those used for reconstitution experiments. They were soluble at approximately 2–3 mg/ml and showed little or no aggregation. These proteins have s⁰_20,w values of 2.0 and 1.5 S, and D_20,w values of 7.6 × 10^?7 and 11.0 × 10^?7 cm² s^?1. Partial specific volumes at 20 °C are 0.730 and 0.740 ml g^?1 for the two proteins. The respective molecular weights determined by sedimentation equilibrium are 24,500 and 12,000. The intrinsic viscosity values for the two proteins are 6.0 and 4.0 ml g^?1. From these hydrodynamic parameters an elongated shape for L3 and a globular shape for L24 have been inferred.     

Interaction of the release factor with the Escherichia coli ribosome: inhibition of the 30S subunit domain by specific antibodies

A domain of the 30S subunit of the Escherichia coli ribosome is in close contact with the release factor when it binds to the 70S particle during the termination of protein biosynthesis. This has been characterised using antibodies specific for the individual proteins of the small ribosomal subunit. Most antibodies do not affect the release factor-mediated reactions but those against S3, S4, S5 and S10 are inhibitory. These proteins are clustered on the lower head and the upper part of the small lobe of the subunit. The regions of these features which are near the interface between the two subunits in the 70S ribosome are known to be close to the base of the stalk of the 50S subunit.     

Further studies on the identity of proteins at the subunit interface of the 70 S E. coli ribosome

The ability of 1 m NH₄Cl to detach iodinated 50 S ribosomal proteins from 50 S subunits and 70 S ribosomes was compared. High salt treatment was effective in preferentially releasing L16, L20, L24, L26, L27, L29, and L30 from the 50 S subunit. Similar but smaller effects were seen for L2, L6, L15, L19, L28, and L31. When these results are combined with several previous studies on accessibility, twelve 50 S proteins appear to be less exposed in the 70 S particle than in the free subunit, by more than one entirely different measure of accessibility. These twelve must be considered strong candidates for possible subunit interface proteins.Lactoperoxidase catalyzed iodination was used to probe the surface topography of active and reversibly inactivated 30 S subunits. The magnesium depleted inactive 30 S particle reproducibly incorporates more ¹²⁵I than the active subunit indicating that a conformational change, characterized by an opening or expansion of the 30 S particles, accompanies 30 S inactivation. Seven 30 S proteins, S5, S21, S4, S7, S10, S13, and S16 become more accessible to lactoperoxidase as a result of inactivation. These proteins are different from those known to become more accessible to lactoperoxidase as a result of the conformational reorganization accompanying subunit association, S3, S6, S9, and S18. Thus, although both inactive 30 S and 50 S-bound 30 S are more open or reactive compared with free active 30 S, the regions which are affected appear to be different.