Binding sites of E. coli and B. stearothermophilus ribosomal proteins on B stearothermophilus 5S RNA.

The primary binding sites for Bacillus stearothermophilus proteins B-L5 and B-L22 and the Escherichia coli proteins E-L5, E-L18 and E-L25 on B. stearothermophilus 5S RNA were determined by limited ribonuclease digestion of the corresponding 5S RNA-protein complexes. The results obtained in this study are in agreement with our previous experiments in which the binding sites of E. coli and B. stearothermophilus proteins were determined for E. coli 5S RNA and lead to the conclusion that the proteins interact with the most conserved regions of 5S RNA. A comparison of the results obtained in this study with those of other published experiments suggest that the proposed interaction of nucleotides 16-21 with those of 58-63 is facilitated by protein binding to 5S RNA.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Cloning, sequencing, and overexpression of genes for ribosomal proteins from Bacillus stearothermophilus

Although a low resolution model for the arrangement of the proteins of the small and large ribosomal subunits is known, a detailed mechanistic understanding of the function of the ribosome awaits a high resolution structure of its components. While crystals have been obtained of several ribosomal proteins from Bacillus stearothermophilus, determination of atomic resolution structures of these proteins is impeded by the difficulty of obtaining large amounts of native proteins for crystallographic or NMR studies. We describe here the cloning and overexpression in Escherichia coli of the genes for ribosomal proteins S5, L6, L9, and L18 from B. stearothermophilus. S5 is extremely toxic to E. coli when overexpressed, and we have taken advantage of a new tightly regulated expression system to obtain high yields (more than 100 mg of pure protein/liter of culture) of this protein. The B. stearothermophilus S5 produced in E. coli crystallizes, and the crystals are identical to those obtained from the native protein. The crystals diffract to 2-A resolution.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Secondary structure of prokaryotic 5S ribosomal ribonucleic acids: a study with ribonucleases

The structures of 5S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus were examined by using ribonucleases A, T1, and T2 and a double helix specific cobra venom ribonuclease. By using both 5' and 3'-32P-end labeling methods and selecting for digested but intact 5S RNA molecules, we were able to distinguish between primary and secondary cutting positions and also to establish the relative degree of cutting. The data reveal the predicted similarities of the higher order structure in the two RNAs but also demonstrate a few significant differences. The data also provide direct evidence for three of the helical regions of the Fox and Woese model of 5S RNA [Fox, G. E., & Woese, C. (1975) Nature (London) 256, 505] and support other important structural features which include a nucleotide looped out from a helical region which has been proposed as a recognition site for protein L18.     

Thermal denaturation of mesophilic and thermophilic 5S ribonucleic acids.

The Tm of Bacillus stearothermophilus 5S ribonucleic acid (RNA) is 1.5 +/- 0.5 C higher than that of 5S RNAs from B. subtilis and Escherichia coli. Melting in 50% methanol and in formaldehyde indicate that both base stacking and helical regions are involved in the slightly increased thermal stability of B. stearothermophilus 5S RNA. It is probable that the 5S RNA makes only a minor contribution to the thermostability of B. stearothermophilus 50S ribosomal subunits.     

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.     

Chemical reactivity of E. coli 5S RNA in situ in the 50S ribosomal subunit.

E. coli 50S ribosomal subunits were reacted with monoperphthalic acid under conditions in which non-base paired adenines are modified to their 1-N-oxides. 5S RNA was isolated from such chemically reacted subunits and the two modified adenines were identified as A73 and A99. The modified 5S RNA, when used in reconstitution of 50S subunits, yielded particles with reduced biological activity (50%). The results are discussed with respect to a recently proposed three-dimensional structure for 5S RNA, the interaction of the RNA with proteins E-L5, E-L18 and E-L25 and previously proposed interactions of 5S RNA with tRNA, 16S and 23S ribosomal RNAs.     

Topography of the C. coli 5S RNA-protein complex as determined by crosslinking with dimethyl suberimidate and dimethyl-3,3''-dithiobispropionimidate

5S RNA-protein complexes were prepared in vitro using partially purified E. coli 5S RNA and total E. coli 70S ribosomal proteins. The complexes were isolated from sucrose gradients and shown to contain proteins L5, L18, L25 and a fourth protein not heretofore characterized and designed L31. The complexes were treated with the crosslinking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate. Both reagents gave identical patterns of crosslinked proteins when analyzed by one-dimensional polyacrylamide/dodecylsulfate gel electrophoresis. Dimers of L5-L31', L5-L18 and L18-L18 and a trimer containing L5, L18 and L31' were identified by diagonal polyacrylamide/dodecylsulfate gel electrophoresis of the proteins crosslinked with dimethyl-3,3'-dithiobispropionimidate. No crosslinking was detected between L25 and the other three proteins.     

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.     

Proteins of the Bacillus stearothermophilus ribosome. The amino acid sequences of proteins S5 and L30

The amino acid sequences of ribosomal proteins S5 and L30 from Bacillus stearothermophilus have been determined. These proteins have recently been crystallized in our institute. Sequence data were obtained by manual sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin and chymotrypsin or thermolysin. Proteins S5 and L30 contain 166 and 62 amino acid residues and have calculated Mr values of 17,628 and 7,053, respectively. Comparison of the sequences with those of the homologous proteins from Escherichia coli shows 55% identical residues for S5 and 53% for L30. For both proteins, the distribution of conserved and substituted regions is not uniform throughout the molecule. Secondary structure predictions were carried out for the B. stearothermophilus proteins. Comparison with the results for the homologous E. coli proteins indicated similar secondary structural order for the molecules from the two species.     

Dynamics of ribosomal RNA structure

The structural dynamics of ribosomal 5S RNAs have been investigated by probing single strandedness through enzymatic cleavage and chemical modification. This comparative study includes 5S rRNAs from E. coli, B. stearothermophilus, T. thermophilus, H. cutirubrum, spinach chloroplast, spinach cytomplasm, and Artemia salina. The structural studies support a unique tertiary interaction in eubacterial 5S rRNAs, involving nucleotides around positions 43 and 75. In addition long range structural effects are demonstrated in E. coli 5S rRNA due to the conversion of C to U at position 92.     

Nucleotide sequences of Bacillus stearothermophilus ribosomal protein genes: part of the ribosomal S10 operon

Restriction fragments from Bacillus stearothermophilus chromosomal DNA were cross-hybridized with the Escherichia coli ribosomal protein L2 gene rplB. A 2-kb EcoRI fragment which showed cross-hybridization was cloned into the M13 phage and sequenced by the dideoxy chain-terminating method. Comparison of the deduced amino-acid sequences with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the region encoding the C-terminus of L2, the genes encoding S19, L22, S3 as well as the N-terminus of L16. Thus the organization of this gene cluster is the same as that in the S10 operon of E. coli. The deduced sequences of proteins L22 and S3, which have not been determined so far, were found to have 52% or 55% amino-acid identity, respectively, with those of the corresponding proteins in E. coli. The deduced B. stearothermophilus S19 protein sequence was in accordance with the reinvestigated protein sequence (H. Hirano, personal communication).     

Methylation of ribosomal proteins in bacteria: evidence of conserved modification of the eubacterial 50S subunit.

Methylation of the 50S ribosomal proteins from Bacillus stearothermophilus, Bacillus subtilis, Alteromonas espejiana, and Halobacterium cutirubrum was measured after the cells were grown in the presence of [1-14C]methionine or [methyl-3H]methionine or both. Two-dimensional polyacrylamide gel electrophoretic analysis revealed, in general, similar relative electrophoretic mobilities of the methylated proteins from each eubacterium studied. Proteins known to be structurally and functionally homologous in several microorganisms were all methylated. Thus, the following group of proteins, which appear to be involved in peptidyltransferase or in polyphenylalanine-synthesizing activity in B. stearothermophilus (P.E. Auron and S. R. Fahnestock, J. Biol. Chem. 256:10105-10110, 1981), were methylated (possible Escherichia coli methylated homologs are indicated in parentheses): BTL5(EL5), BTL6(EL3), BTL8(EL10), BTL11(EL11), BTL13(EL7L12) and BTL20b(EL16). In addition, the pentameric ribosomal complex BTL13 X BTL8, analogous to the complex EL7L12 X EL10 of E. coli, contained methylated proteins. Analysis of the methylated amino acids in the most heavily methylated proteins, BSL11 from B. subtilis and BTL11 from B. stearothermophilus, showed the presence of epsilon-N-trimethyllysine as the major methylated amino acid in both proteins, in agreement with known data for E. coli. In addition, BSL11 appeared to contain trimethylalanine, a characteristic, modified amino acid previously described only in EL11 from E. coli. These results and those previously obtained from other bacteria indicate a high degree of conservation for ribosomal protein methylation and suggest an important, albeit unknown, role for the modification of these components in eubacterial ribosomes.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.     

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.     

Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis.

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D.