Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

Sequence Studies on Precursor 16S Ribosomal RNA of <Emphasis Type="Italic">Escherichia coli</Emphasis>

THE 16S and 23S rRNAs of E. coli are known to be synthesized through intermediates of marginally. higher S values (about 17S and 24S). Although various authors^1–3 have reported the presence of these precursors, no sequence studies have been presented. Nevertheless, there is strong but indirect evidence³ that the precursor 16S rRNA (p16) is about 200 residues longer than 16S rRNA (ml6). In contrast, the evidence suggests that the precursor 23S rRNA has few or no further residues.     

RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA

Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S¹ RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S¹ RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S¹ RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S¹ RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T₁ ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg²⁺ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Long tail fibres of the novel broad‐host‐range T‐even bacteriophage S16 specifically recognize Salmonella OmpC

We report isolation and characterization of the novel T4‐like Salmonella bacteriophage vB_SenM‐S16. S16 features a T‐even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full‐length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects ‘rough’ Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.     

Comparison of 16S rRNA gene sequences of genus <Emphasis Type="Italic">Methanobrevibacter</Emphasis>

Background  

The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.     

The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50S subunits causes an apparent increase in the S21-16S RNA and S21-S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1-16S-RNA distance.     

Prediction of secondary structures of 16S and 23S rRNA fromEscherichia coli

Small and large subunits ofEscherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared Briefly with earlier model Building studies.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.     

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts: Recombinant plasmid; sequence homology; stem and loop structure

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts has been determined. This nucleotide sequence has 96% homology with that of maize chloroplast 16S rRNA gene and 74% homology with that of Escherichia coli16S gene.The 3′ terminal region of this gene contains the sequence ACCTCC which is complementary to sequences found at the 5′ termini of prokaryotic mRNAs.The large stem and loop structure can be constructed from the sequences surrounding the 5′ and 3′ ends of the 16S gene. These observations demonstrate the prokaryotic nature of chloroplast 16S rRNA.     

Protein binding sites on Escherichia coli 16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9.

14C-labelled proteins from E. coli 30S ribosomal subunits were isolated by HPLC, and selected groups of these proteins were reconstituted with 32P-labelled 16S RNA. The isolated reconstituted particles were partially digested with ribonuclease A, and the RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Protein S7 alone gave no protected fragments, but S7 together with S14 and S19 protected an RNA region comprising the sequences 936-965, 972-1030, 1208-1262 and 1285-1379 of the 16S RNA. Addition of increasing amounts of protein S9 to the S7/S14/S19 particle resulted in a parallel increase in the protection of the hairpin loop between bases 1262 and 1285. The results are discussed in terms of the three-dimensional folding of 16S RNA in the 30S subunit.     

Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease

A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. ³²P-labeled 30 S subunits or reconstituted 70 S tight couples containing ³²P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.     

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.