Translational regulation by ribosomal protein S8 in Escherichia coli: structural homology between rRNA binding site and feedback target on mRNA.

It has been previously shown that ribosomal protein synthesis in Escherichia coli is regulated at the level of translation by certain key ribosomal proteins. In the spc operon, S8 regulates the expression of L5 and some of the subsequent genes, while the first two genes (L14 and L24) are regulated independently. We therefore determined the DNA sequence at the junction of the L24 and L5 genes, which corresponds to the putative feedback target for S8. We show that there is a striking homology between the structure of the mRNA for this region and the known binding site for S8 on 16S rRNA. These results support the theory that the regulation of ribosomal protein synthesis is based on competition between rRNA and mRNA for regulatory ribosomal proteins.     

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.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon.

The structural features of Escherichia coli ribosomal protein S8 that are involved in translational regulation of spc operon expression and, therefore, in its interaction with RNA have been investigated by use of a genetic approach. The rpsH gene, which encodes protein S8, was first inserted into an expression vector under the control of the lac promoter and subsequently mutagenized with methoxylamine or nitrous acid. A screening procedure based on the regulatory role of S8 was used to identify mutants that were potentially defective in their ability to associate with spc operon mRNA and, by inference, 16S mRNA. In this way, we isolated 39 variants of the S8 gene containing alterations at 34 different sites, including 37 that led to single amino acid substitutions and 2 that generated premature termination codons. As the mutations were distributed throughout the polypeptide chain, our results indicate that amino acid residues important for the structural integrity of the RNA-binding domain are not localized to a single segment. Nonetheless, the majority were located within three short sequences at the N terminus, middle, and C terminus that are phylogenetically conserved among all known eubacterial and chloroplast versions of this protein. We conclude that these sites encompass the main structural determinants required for the interaction of protein S8 with RNA.     

The structure of a ribosomal protein S8/spc operon mRNA complex

In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.     

Computer analysis of regulatory signals in complete bacterial genomes. Translation initiation of ribosomal protein operons]

Signals of translation initiation of operons of Haemophilus influenzae ribosomal proteins were predicted. This process is regulated by the formation of secondary RNA structures to which one of the proteins encoded in a particular operon binds. In some cases, these structures imitate the region of protein binding to rRNA. Predictions are made by comparing with homologous operons of Escherichia coli and analogous regions of rRNA and by estimating the energy of secondary structure formation. It is shown that this regulatory mechanism occurs: in operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium.     

The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and hydroxyl radical probing studies.

The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules. The S8 binding site on 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means. We performed in vitro iterative selection of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified. These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution. Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642). The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important. Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.     

Selection of random RNA fragments as method for searching for a site of regulation of translation of <Emphasis Type="Italic">E. coli</Emphasis> streptomycin mRNA by ribosomal protein S7

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.     

A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.     

Analysis of the spc ribosomal protein operon of Thermus aquaticus

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.     

Feedback regulation of ribosomal protein synthesis in E. coli: Localization of the mRNA target sites for repressor action of ribosomal protein L1

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins from a single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.