Identification of protein S 1 at the messenger RNA binding site of the Escherichia coli ribosome

Poly-4-thiouridylic acid acts as messenger RNA for polyphenylalanine synthesis in an $\text{in vitro}$ protein synthesizing system. When a complex consisting of ribosomes, poly-4-thiouridylic acid and Phe-tRNA is irradiated at 300 to 400 nm, covalent bonds between this messenger RNA and protein S 1 are formed.     

Iodination of Escherichia coli ribosomal protein L18 abolishes its 5 S RNA binding activity

Iodination of Escherichia coli ribosomal protein L18 inactivated the 5 S RNA binding activity of the protein. Complete activity loss occurred at a 4-fold molar excess of iodine to L18. Tyrosine was found to be the reactive amino acid. L18, prebound to 5 S RNA, was inactivated at a much slower rate than unbound L18. Treatment of L18 with tetranitromethane also resulted in an inactivation of the protein. However, much larger amounts of tetranitromethane, compared to iodine, were necessary to achieve inactivation (50% activity loss at a 600-fold molar excess of tetranitromethane to L18).     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli

Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1. A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11. The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests. It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E. coli 23 S rRNA.     

Exploration of the L18 binding site on 5S RNA by deletion mutagenesis.

Several deletion variants of E. coli 5S RNA have been constructed and produced either in vivo or in vitro using T7 RNA Polymerase. Their structures and ribosomal protein L18 binding properties have been examined. All of them are similar to wild-type 5S RNA in their helix II-III regions, where L18 binds [Huber, P.W. and Wool, I.G. (1984) Proc. Natl. Acad. Sci. (USA) 81, 322-326; Douthwaite, S., Christensen, A., and Garrett, R.A. (1982) Biochemistry 21, 2313-2320.], by NMR criteria. However, none of the molecules examined that lack the helix IV-helix V stem bind L18 efficiently, even though that portion of 5S RNA is outside the L18 footprint. The L18 binding site is clearly more than a simple hairpin loop.     

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.     

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)     

Sequence determination of protein S9 from the Escherichia coli ribosome.

The complete amino acid sequence of ribosomal protein S9 of Escherichia coli has been established. The protein was digested with trypsin and Staphylococcus aureus protease and the resulting peptides were separated by ion exchange chromatography on a new Dowex 50W-X7 microcolumn or a small phosphocellulose column. If necessary, they were rechromatographed on purified cellulose thin-layer plates on a preparative scale. The sequences of the peptides were determined by the micro dansyl-Edman technique, whereas the alignments of the tryptic peptides were mainly established from large cyanogen bromide fragments which were sequenced by the automatic Edman degradation process. Protein S9 is 128 amino acids long and has the following composition: Asx7, Thr5, Ser7, Glx16, Pro3, Gly13, Ala10, Val10, Met3, Ile7, Leu9, Tyr5, Phe4, His1, Lys10, Arg18. The molecular weight as calculated from the amino acid composition is 14 569. A total of 92.6 mg of the lyophilized protein was used for the determination of the primary structure of S9. Most of the material was needed to isolate sufficient amounts of the CNBr-fragments for the automatic degradation in the sequenator.     

The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli

During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.     

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.

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.     

A functional hybrid ribosome binding site in tryptophan operon messenger RNA of Escherichia coli

We present evidence that repair of DNA damage induced by decay of incorporated ¹²⁵I after replication of the labeled duplex of Escherichia coli requires the recA⁺ gene function. Furthermore, only about half of the cells survive after label segregation even when that repair function is present. Our results support the possibility that repair of ¹²⁵I decay-induced lesions is asymmetric, being limited to damage initiated in only one of the two strands of the DNA duplex.     

The mechanism of covalent reaction of bromoacetyl-phenylalanyl-transfer RNA with the peptidyl-transfer RNA binding site of the Escherichia coli ribosome

Results are presented to prove that bromoacetyl-phenylalanyl-transfer RNA reacts covalently with 50 S ribosomal proteins L2 and L27 while it is bound correctly to the peptidyl site on the 70 S ribosome. Attachment of the BrAcPhe moiety to tRNA causes a 100-fold enhancement of its reactivity with ribosomes. This reactivity closely parallels binding of tRNA whether measured by poly(U) stimulation or competition with deacylated tRNA. BrAcPhe-tRNA can bind correctly to the P site as judged by puromycin releasibility and lack of tetracycline inhibition. Little significant reaction of BrAcPhe-tRNA with L2 and L27 occurs during procedures used to purify and analyze ribosomal proteins. If ribosomes are first incubated with BrAcPhe-tRNA and subsequently treated with puromycin before analysis, little inhibition of the covalent reaction with L2 and L27 is observed. In contrast, a few minor reaction products are markedly suppressed. Covalently attached BrAcPhe-tRNA is still capable of accepting an amino acid from Phe-tRNA or puromycin. The products from this reaction are found attached to proteins L2 and L27 and to a lesser extent to L15 and L16. This shows that true affinity labeling of proteins in the peptidyl binding site has been accomplished.Some covalent reaction of BrAcPhe-tRNA with the 30 S protein S18 is also observed. This reaction is not poly(U)-dependent, however, and S18-reacted BrAcPhe-tRNA is not capable of peptide bond formation with Phe-tRNA. It seems likely that reaction with S18 results from a non-functional interaction of the affinity label with the ribosome.     

Small-angle X-ray titration study on the complex formation between 5-S RNA and the L18 protein of the Escherichia coli 50-S ribosome particle.

The 5-S RNA (A) and the L18 protein (B) from Escherichia coli ribosomes form one single AB complex in the concentration ranges supposed to prevail in vivo; at concentrations of L18 higher than 40 mM there is some indication for a minor species, most probably an AB2 species. This is indicated from the X-ray scattering titration data of the 5-S RNA/L18 system recorded at 21 degrees C in ribosomal reconstitution buffer. As a result of the 1:1 complex formation, there is a relatively small but defined increase in the radius of gyration from 3.61 to 3.85 nm. This result as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L18 model is quite far from the electron density centre and where protein L18 interacts with one or both of the minor arms of the supposed Y-shaped 5-S RNA molecule.