Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Chemical accessibility of 18S rRNA in native ribosomal complexes: interaction sites of mRNA, tRNA and translation factors

During protein synthesis the ribosome interacts with ligands such as mRNA, tRNA and translation factors. We have studied the effect of ribosome-ligand interaction on the accessibility of 18S rRNA for single strand-specific modification in ribosomal complexes that have been assembled in vivo, i. e. native polysomes. A comparison of the modification patterns derived from programmed and non-programmed ribosomes showed that bases in the 630- and 1060-loops (530- and 790-loops in E. coli) together with two nucleotides in helices 33 and 34 were protected from chemical modification. The majority of the protected sites were homologous to sites previously suggested to be involved in mRNA and/or tRNA binding in prokaryotes and eukaryotes, implying that the interaction sites for these ligands are similar, if not identical, in naturally occurring programmed ribosomes and in in vitro assembled ribosomal complexes. Additional differences between programmed and non-programmed ribosomes were found in hairpin 8. The bases in helix 8 showed increased exposure to chemical modification in the programmed ribosomes. In addition, structural differences in helices 36 and 37 were observed between native 80S run-off ribosomes and 80S ribosomes assembled from isolated 40S and 60S subunits.     

Interaction of ribosomal protein S1 and initiation factor IF3 with the 3' major domain and the decoding site of the 30S subunit of Escherichia coli.

We have studied the effect of the binding of ribosomal protein S1 and initiation factor IF3 on the accessibility of nucleotide residues 584-1506 in the small subunit of the Escherichia coli ribosome. Protein S1 strongly decreases RNase V1 attack at G1164, in hairpin 40 of the 3' major domain, and weakly decreases DMS attack at C1302, in the central loop of the 3' major domain, and at A1503, in the 3' minor domain. It also weakly increases the DMS reactivity of A1004, in the 3' major domain, and of A901, in the central domain. Factor IF3 strongly decreases RNase V1 attack (but not dimethyl sulfate attack) at A1408, in the decoding site, and weakly protects A1500, in the 3' minor domain and near the colicin E3 cleavage site. Neomycin does not interfere with this effect of IF3, but IF3 interferes with the protective effect of neomycin against dimethyl sulfate attack at A1408.     

Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation.     

Structural analysis of kasugamycin inhibition of translation

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).     

Binding sites for ribosomal proteins S8 and S15 in the 16 S RNA of Escherichia coli.

A fragment of the 16 S ribosomal RNA of Escherichia coli that contains the binding sites for proteins S8 and S15 of the 30 S ribosomal subunit has been isolated and characterized. The RNA fragment, which sediments as 5 S, was partially protected from pancreatic RNAase digestion when S15 alone, or S8 and S15 together, were bound to the 16 S RNA. Purified 5 S RNA was shown to reassociate specifically with protein S15 by analysis of binding stoichiometry. Although interaction between the fragment and protein S8 alone could not be detected, the 5 S RNA selectively bound both S8 and S15 when incubated with an unfractionated mixture of 30-S subunit proteins. Nucleotide sequence analysis demonstrated that the 5 S RNA arises from the middle of the 16 S RNA molecule and encompasses approximately 150 residues from Sections C, C'1 and C'2. Section C consists of a long hairpin loop with an extensively hydrogen-bonded stem and is contiguous with Section C'1. Sections C'1 and C'2, although not contiguous, are highly complementary and it is likely that together they comprise the base-paired stem of an adjacent loop.     

Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy

BACKGROUND: Ribosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy. RESULTS: A tRNA sequence inserted into the E. coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral 'foot' structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined. CONCLUSIONS: Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.     

Protection of ribosomal RNA from kethoxal in polyribosomes: Implication of specific sites in ribosome function

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specifie reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.     

Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.     

Prediction of three-dimensional structure of Escherichia coli ribosomal RNA

A model for the tertiary structure of 23S, 16S and 5S ribosomal RNA molecules interacting with three tRNA molecules is presented using the secondary structure models common to E. coli, Z. mays chloroplast, and mammalian mitochondria. This ribosomal RNA model is represented by phosphorus atoms which are separated by 5.9 A in the standard A-form double helix conformation. The accumulated proximity data summarized in Table 1 were used to deduce the most reasonable assembly of helices separated from each other by at least 6.2 A. Straight-line approximation for single strands was adopted to describe the maximum allowed distance between helices. The model of a ribosome binding three tRNA molecules by Nierhaus (1984), the stereochemical model of codon-anticodon interaction by Sundaralingam et al. (1975) and the ribosomal transpeptidation model, forming an alpha-helical nascent polypeptide, by Lim & Spirin (1986), were incorporated in this model. The distribution of chemically modified nucleotides, cross-linked sites, invariant and missing regions in mammalian mitochondrial rRNAs are indicated on the model.     

Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension

We have examined the effect of binding ribosomal protein S4 to 16 S rRNA on the susceptibility of the RNA to a variety of chemical and enzymatic probes. We have used dimethyl sulfate to probe unpaired adenines (at N-1) and cytosines (at N-3), kethoxal to probe unpaired guanines (at N-1 and N-2) and cobra venom (V1) ribonuclease as a probe of base-paired regions of 16 S rRNA. Sites of attack by the probes were identified by primer extension using synthetic oligodeoxynucleotides. Comparison of probing results for naked and S4-bound rRNA shows: Protein S4 protects a relatively compact region of the 5' domain of 16 S rRNA from chemical and enzymatic attack. This region is bounded by nucleotides 27 to 47 and 394 to 556, and has a secondary structure characterized by the junction of five helical elements. Phylogenetically conserved irregular features (bulged nucleotides, internal loops and flanking unpaired nucleotides) and helical phosphodiester bonds of four of the helices are specifically protected in the S4-RNA complex. We conclude that this is the major, and possibly sole region of contact between 16 S rRNA and S4. Many of the S4-dependent changes mimic those observed on assembly of 16 S rRNA into 30 S ribosomal subunits. Binding of S4 causes enhanced chemical reactivity coupled with protection from V1 nuclease outside the S4 junction region in the 530, 720 and 1140 loops. We interpret these results as indicative of loss of structure, and suggest that S4 binding causes disruption of adventitious pairing in these regions, possibly by stabilizing the geometry of the RNA such that these interactions are prevented from forming.     

Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements.

Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.     

Real-time evidence for EF-G-induced dynamics of helix 44 in 16S rRNA

The penultimate stem-loop of 16S ribosomal RNA (rRNA), helix 44, plays a central role in ribosome function. Using time-resolved dimethyl sulfate (DMS) probing, we have analyzed time-dependent modifications that occur at specific bases in this helix near the decoding region, resulting from the binding of elongation factor G (EF-G) in various forms. When EF-G-GTP is bound to 70S ribosomes, bases A1492 and A1493 are immediately protected, while other bases in the region show either no change or enhanced modification. When apo-EF-G is bound to 70S ribosomes and GTP is added, substantial transient time-dependent enhancement occurs at bases A1492 and A1493, with somewhat less enhancement occurring at base A1483, all in the first 45 ms. When mRNA and deacylated tRNAs are bound to the 70S ribosome and EF-G-GTP is added, bases A1492 and A1493 again show substantial and continued enhancement, while bases A1408, A1413, and A1418 all show time-dependent protection. These results provide primary, real-time evidence that EF-G induces direct or indirect structural changes in this region as EF-G is bound and as GTP is hydrolyzed.     

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Antibiotic interactions at the GTPase-associated centre within Escherichia coli 23S rRNA.

A comprehensive range of chemical reagents and ribonucleases was employed to investigate the interaction of the antibiotics thiostrepton and micrococcin with the ribosomal protein L11-23S RNA complex and with the 50S subunit. Both antibiotics block processes associated with the ribosomal A-site but differ in their effects on GTP hydrolysis, which is inhibited by thiostrepton and stimulated by micrococcin. The interaction sites of both drugs were shown to occur within the nucleotide sequences A1067-A1098 within the protein L11 binding site on 23S RNA. This region of the ribosome structure is involved in elongation factor-G-dependent GTP hydrolysis and in the stringent response. No effects of drug binding were detected elsewhere in the 23S RNA. In general, the two drugs afforded 23S RNA similar protection from the chemical and nuclease probes in accord with their similar modes of action. One important exception, however, occurred at nucleotide A1067 within a terminal loop where thiostrepton protected the N-1 position while micrococcin rendered it more reactive. This difference correlates with the opposite effects of the two antibiotics on GTPase activity.