Probing dynamic changes in rRNA conformation in the 30S subunit of the Escherichia coli ribosome.

Ribosomal RNA molecules within each ribosomal subunit are folded in a specific three-dimensional form. The accessibility of specific sequences of rRNA of the small ribosomal subunit of Escherichia coli was analyzed using complementary oligodeoxyribonucleotides, 6-15 nucleotides long. The degree of hybridization of these oligomers to their RNA complements within the 30S subunit was assessed using nitrocellulose membrane filter binding assays. Specifically, the binding of short DNA oligomers (hexameric and longer) complementary to nucleotides 919-928, 1384-1417, 1490-1505, and 1530-1542 of 16S rRNA was monitored, and in particular how such binding was affected by the change in the activation state of the subunit. We found that nucleotides 1397-1404 comprise an unusually accessible sequence in both active and inactive subunits. Nucleotides 919-924 are partially available for hybridization in active subunits and somewhat more so in inactive subunits. Nucleotides 1534-1542 are freely accessible in active, but only partially accessible in inactive subunits, while nucleotides 1490-1505 and 1530-1533 are inaccessible in both, under the conditions tested. These results are in general agreement with results obtained using other methods and suggest a significant conformational change upon subunit activation.     

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.     

Localization of the site of cleavage of ribosomal RNA by colicin E3. Placement on the small ribosomal subunit by electron microscopy of antibody--complementary oligodeoxynucleotide complexes

Colicin E3 is a ribonuclease that inactivates Escherichia coli ribosomes by cleaving the RNA of the small ribosomal subunit after nucleotide 1493. A series of oligodeoxynucleotides that complement 16 S RNA in the region of the colicin cleavage site has been synthesized, and their ability to form complexes with 30 S ribosomal subunits has been measured using a nitrocellulose filter-binding assay. The most efficiently bound probe, complementary to residues 1485-1496, was modified with antibody-recognizable derivatives at the 5'-end, the 3'-end, or both. Antibody-oligonucleotide-subunit complexes were then generated and examined by electron microscopy. Antibody binding was seen at the tip of the platform of the 30 S subunit. The complementary oligonucleotide and thus the site at which colcin E3 cleavage occurs is therefore in the same physical region as the 3'-end of the 16 S ribosomal RNA and its message-positioning "Shine-Dal-garno" sequence.     

Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action.     

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.     

Localization of an oligodeoxynucleotide complementing 16S ribosomal RNA residues 520-531 on the small subunit of Escherichia coli ribosomes: electron microscopy of ribosome-cDNA-antibody complexes.

The oligodeoxynucleotide dACCGCGGCTGCT, complementary to Escherichia coli small ribosomal subunit RNA residues 520-531, has been used to probe subunit conformation and to localize the sequence in the subunit. Conditions for binding of the cDNA to 30S subunits were optimized and specificity of the interaction was demonstrated by RNase H cleavage. Three kinds of terminal modification of this cDNA were used to allow its localization by immune electron microscopy. A solid phase support with 5'-dimethoxytrity-N6-delta 2-isopentenyl-adenosine linked to controlled pore glass was synthesized, and used to prepare oligomer with an added 3'-terminal residue of isopentenyl adenosine. cDNA with a 5' primary amine substituent was modified with 1-fluoro-2,4-dinitrobenzene to prepare 5'-dinitrophenyl oligonucleotide, and both modifications together gave doubly-derivatized probes. Immune electron microscopy with antibodies to dinitrophenol, isopentenyl adenosine, or both, was used to place the cDNA on 30S subunits. In each case the probe was placed at a single site at the junction of the head and body of the subunit, near the decoding site and the area in which elongation factor Tu is bound. It is proposed that this segment of ribosomal RNA functions in mRNA binding and orientation.     

Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy

The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site. High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence. Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base. These antibodies were reacted with 30 S ribosomal subunits from E. coli and from the chloroplasts of Alaskan peas. These two types of ribosome have been shown to be topographically similar (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers. In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform. The analogous localization in both E. coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin.     

Localization of a segment of 16S RNA on the surface of the small ribosomal subunit by immune electron microscopy of complementary oligodeoxynucleotides.

Oligonucleotides that complement Escherichia coli 16S ribosomal RNA residues 685-696 and 694-705 have been synthesized so as to incorporate antibody-recognizable markers: a 3'-terminal residue of N6-delta 2-isopentenyladenosine, a 5'-dinitrophenyl group, or both. Each oligonucleotide is able to bind RNA within the small ribosomal subunit, whether free or in 70S ribosomes. Immune electron microscopy places probes at nucleotides 685, 694 and 705 within a single area, at the tip of the subunit platform, very near the position of the 3'-end of the 16S RNA.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.     

An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states

HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.     

Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, GFR.

It is well established that when E. coli 30S ribosomal subunits are irradiated with ultraviolet light under mild conditions a specific cross-link is formed between protein S7 and the 16S RNA. Methodology is presented for the analysis of the single nucleotide residue concerned in this cross-link. Firstly, the identity of the ribonuclease T1 octanucleotide attached to S7 is confirmed by a new method, which involves isolation and analysis of S7-polynucleotide complexes containing 30 -- 40 nucleotides. Secondly, the isolated S7-octanucleotide complex is digested successively with ribonuclease A, proteinase K and ribonuclease T2, and the nucleotides liberated are identified. The results show unambiguously that uridine residue number 1239 in the 16S RNA sequence is cross-linked to protein S7.     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro.

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.     

Identification of several proteins involved in the messenger RNA binding site of the 30 S ribosome by inactivation with 2-methoxy-5-nitrotropone

Chemical modification of unwashed 30 S ribosomal subunits with 2-methoxy-5-nitrotropone causes a rapid loss of their capacity to bind bacteriophage Qβ RNA. Reconstitution experiments show that ribosomal protein is the functionally inactivated species. When purified unmodified ribosomal proteins were included in a mixture of 16 S ribosomal RNA and total protein derived from 2-methoxy-5-nitrotropone-treated subunits, four proteins (S1, S12, S13 and S21) were found to promote the reconstitution of particles capable of binding natural messenger RNA.     

Mechanism of ribosomal subunit association: discrimination of specific sites in 16 S RNA essential for association activity.

Modification of 30 S ribosomal subunits with kethoxal causes loss of their ability to associate with 50 S subunits under tight couple conditions. To identify those 16 S RNA sequences important for the association. ³²P-labeled 30 S subunits were partially inactivated by reaction with kethoxal. The remaining association-competent 30 S subunits were selected from the modified population by their ability to form 70 S ribosomes. Comparison of kethoxal diagonal maps of the association-competent subunits with those of the total population of modified subunits reveals nine sites in 16 S RNA whose modification leads to loss of association activity. Eight of these sites were previously found to be protected from kethoxal attack and one was shown to have enhanced reactivity in 70 S ribosomes (Chapman &; Noller, 1977). As before, these sites are not distributed thoughout the molecule, but are found to be clustered in two regions, at the middle and at the 3′ terminus of the 16 S RNA chain.We interpret these findings in terms of a simple preliminary model for the functional organization of 16 S RNA, supported by the observations of other investigators, in which we divide the molecule into four domains. (1) Residues 1 to 600 are involved mainly in structural organization and assembly. (2) Residues 600 to 850 include sites which make contact with the 50 S subunit and are essential for subunit association. (3) Sites from the domain comprising residues 850 to 1350 line a pocket at the interface between the two ribosomal subunits. and contribute to the binding site(s) for transfer RNA. (4) Residues 1350 to 1541 also contain sequences which bind the 50 S subunit, but some sites in this domain alternatively participate in the initiation of protein synthesis.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.