Domain Of 16S rRNA Research Articles

Molecular Phylogenetic Analysis of 16S rRNA Sequences Identified Two Lineages of Helicobacter pylori Strains Detected from Different Regions in Sudan Suggestive of Differential Evolution.

Background Helicobacter pylori (H. pylori) is ubiquitous among humans and one of the best-studied examples of an intimate association between bacteria and humans. Phylogeny and Phylogeography of H. pylori strains are known to mirror human migration patterns and reflect significant demographic events in human prehistory. In this study, we analyzed the molecular evolution of H. pylori strains detected from different tribes and regions of Sudan using 16S rRNA gene and the phylogenetic approach. Materials and methods. A total of 75 gastric biopsies were taken from patients who had been referred for endoscopy from different regions of Sudan. The DNA extraction was performed by using the guanidine chloride method. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied, and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Results Molecular detection of H. pylori has shown that 28 (37.33%) of the patients were positive for H. pylori and no significant differences were found in sociodemographic characteristics, endoscopy series, and H. pylori infection. Nucleotide variations were observed at five nucleotide positions (positions 219, 305, 578, 741, and 763–764), and one insertion mutation (750_InsC_751) was present in sixty-seven percent (7/12) of our strains. These six mutations were detected in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding sites; 66.67% of them were located in the central domain of 16S rRNA. The phylogenetic analysis of 16S rRNA sequences identified two lineages of H. pylori strains detected from different regions in Sudan. The presence of Sudanese H. pylori strains resembling Hungarian H. pylori strains could reflect the migration of Hungarian people to Sudan or vice versa. Conclusion This finding emphasizes the significance of studying the phylogeny of H. pylori strains as a discriminatory tool to mirror human migration patterns. In addition, the 16S rRNA gene amplification method was found useful for bacterial identification and phylogeny.

Mutations Preventing the Phosphorylation of Human Ribosomal Protein uS15 at Y38 and S48 Reduce the Efficiency of its Transfer into the Nucleolus

The ribosomal protein uS15 is one of the key proteins forming the small (40S) ribosomal subunit structure around the central domain of 18S rRNA. According to a number of proteomic studies based on mass-spectrometry analysis, there are many phosphorylation sites in this protein. However, when the protein uS15 is contained in the 40S subunit, it does not carry such posttranslational modification. In this study, it was found that the cytoplasmic protein kinases of HEK293T cells are able to phosphorylate the human recombinant ribosomal protein uS15 with significantly greater efficiency than that of the nucleus protein kinases. The effect of the amino acid substitutions Y38A and S48A preventing the phosphorylation of the ribosomal protein uS15 at the corresponding sites, on the transport of the recombinant protein uS15 fused to the green fluorescent protein in the nucleolus was studied. It was shown that single mutations at the above sites have little effect on the transport of this protein, whereas double mutation reduces the efficiency of this process by more than a quarter. The findings suggest the importance of phosphorylation of the ribosomal protein uS15 by cytoplasmic protein kinases at several sites, including Y38A and S48A, for its efficient transfer into the nucleolus, where pre-ribosomal subunits are assembled.

Structural and functional analysis of ribosome assembly factor Efg1.

Ribosome biogenesis in eukaryotes is a complicated process that involves association and dissociation of numerous assembly factors and snoRNAs. The yeast small ribosomal subunit is first assembled into 90S pre-ribosomes in an ordered and dynamic manner. Efg1 is a protein with no recognizable domain that is associated with early 90S particles. Here, we determine the crystal structure of Efg1 from Chaetomium thermophilum at 3.3 Å resolution, revealing a novel elongated all-helical structure. Efg1 is not located in recently determined cryo-EM densities of 90S likely due to its low abundance in mature 90S. Genetic analysis in Saccharomyces cerevisiae shows that the functional core of Efg1 contains two helical hairpins composed of highly conserved residues. Depletion of Efg1 blocks 18S rRNA processing at sites A1 and A2, but not at site A0, and production of small ribosomal subunits. Efg1 is initially recruited by the 5′ domain of 18S rRNA. Its absence disturbs the assembly of the 5′ domain and inhibits release of U14 snoRNA from 90S. Our study shows that Efg1 is required for early assembly and reorganization of the 5′ domain of 18S rRNA.

Co-Transcriptional Ribosome Assembly in Real-Time

The bacterial ribosome is a large macromolecular machine composed of three large RNA molecules and more than 50 proteins, which are co-transcriptionally assembled in a living cell within 1-2 minutes.The molecular mechanism of assembly has extensively been studied with pre-transcribed ribosomal RNA (rRNA) mainly using ensemble methods, in which binding of each ribosomal protein (r-protein) is detected as the average state of all populations in which it is present. Because active transcription at the correct speed is essential for ribosome assembly at physiological conditions, the molecular mechanism of ribosome assembly has to be understood co-transcriptionally.Here we present a novel single-molecule fluorescence based approach in which transcription and folding of single rRNA molecules and the binding of r-proteins to them can be visualized simultaneously in real-time.Using zero-mode waveguide instrumentation, we could monitor transcription progression of single RNA molecules of several hundred nucleotides.Studying the co-transcriptional binding of the primary r-protein S15 to the central domain shows that besides short lived binding events (few seconds lifetime), specific S15 binding (>30 seconds lifetime) can occur shortly after transcription of its binding site. In contrast, co-transcriptional binding of primary protein S7 to the entire 3’-domain is much more dynamic with some RNA molecules being unable to stably bind S7 (seconds lifetime) while others can eventually bind S7 specifically, yet can do so only minutes after transcription at 20 degrees Celsius.Overall, using a novel approach to observe in real-time binding of single r-proteins to transcribing r-RNA, we see that co-transcriptional folding of the central 16S rRNA domain is efficient allowing immediate protein binding, while 3’-domain folding is inefficient, indicating that other r-proteins or assembly factors such as RNA helicases must increase folding efficiency in the cell (at least at low temperature).

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles-S3, S10, S14, and S21-all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increased levels of precursor 17S rRNA in assembly intermediates. Mutation ΔlepA confers a synthetic growth phenotype in absence of RsgA, another GTPase, well known to act in 30S subunit assembly. Analysis of the ΔrsgA strain reveals accumulation of intermediates that resemble those seen in the absence of LepA. These data suggest that RsgA and LepA play partially redundant roles to ensure efficient 30S assembly.

Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3′-terminal domain

Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3′-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA–protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed.

F-108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs.

Ecological studies of microbial communities often use profiling methods but the true community diversity can be underestimated in methods that separate amplicons based on sequence length using performance optimized polymer 4. Taxonomically, unrelated organisms can produce the same length amplicon even though the amplicons have different sequences. F-108 polymer has previously been shown to resolve same length amplicons by sequence polymorphisms. In this study, we showed F-108 polymer, using the ABI Prism 310 Genetic Analyzer and CE, resolved four bacteria that produced the same length amplicon for the 16S rRNA domain V3 but have variable nucleotide content. Second, a microbial mat community profile was resolved and supported by NextGen sequencing where the number of peaks in the F-108 profile was in concordance with the confirmed species numbers in the mat. Third, equine DNA was analyzed for SNPs. The F-108 polymer was able to distinguish heterozygous and homozygous individuals for the melanocortin 1 receptor coat color gene. The method proved to be rapid, inexpensive, reproducible, and uses common CE instruments. The potential for F-108 to resolve DNA mixtures or SNPs can be applied to various sample types-from SNPs to forensic mixtures to ecological communities.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states.

The three-dimensional conformations of noncoding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a 16S rRNA domain for which SHAPE (selective 2′-hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo- and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M2) experiments highlight issues of prior methodology and instead give a near-crystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M2-based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.

The novel mitochondrial 16S rRNA 2336T>C mutation is associated with hypertrophic cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) is a primary disorder characterised by asymmetric thickening of septum and left ventricular wall, with a prevalence of 0.2% in the general population.ObjectiveTo describe a novel mitochondrial...

Mg2+ ions affect structure of central domain of 18S rRNA near ribosomal protein S13 binding site

At high concentrations, Mg2+ ions are known to stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. The effect of Mg2+ on the formation of the 18S rRNA structure during 40S subunit assembly remains poorly explored. In this work, we show that a sequential increase in the Mg2+ concentration from 0.5 to 20 mM leads to a significant decrease in the affinity of recombinant human ribosomal protein S13 (rpS13e) to the RNA transcript that corresponds to the central domain fragment of 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD, including the nucleotides of helices H20 and H22, the availability of which to hydroxyl radicals depended on the Mg2+ concentration, were determined. The increase in the Mg2+ concentration was shown to result in the enhanced accessibility of nucleotides G933–C937 and C1006–A1009 in helix H22 and reduced accessibility of nucleotides A1023, A1024, and C1026–A1028 in the H20 helix. A comparison of the obtained results with crystallographic data of the central domain of 18S rRNA in the 40S ribosomal subunit of Saccharomyces cerevisiae has led to the conclusion that an increase in the Mg2+ concentration resulted in the reorientation of helices H20 and H24 relative to to helices H22 and H23 and the formation of a structure in which these helices were positioned the same way as in 40S subunit. Hence, the saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding, and results in the decreased affinity of this domain to the protein.

Ионы Mg2+влияют на структуру центрального домена 18S рРНК вблизи участка связывания рибосомного белка S13

It is known that Mg2+ ions at high concentrations stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. Effect of Mg2+ on the formation of the 18S rRNA structure at the 40S subunit assembly remains poorly explored. In this paper, we show that the sequentional increase of the Mg2+ concentration from 0.5 mM to 20 mM leads to a significant decrease of the affinity of recombinant human ribosomal protein S13 (rpS13e) to a RNA transcript corresponding to the central domain fragment of the 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD (including the nucleotides of helices H20 and H22), whose availabilities to hydroxyl radicals were dependent on the Mg2+ concentration, were determined. It was found that increase of the concentrations of Mg2+ results in the enhanced accessibilities of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduces those of nucleotides A1023, A1024, and A1028-S1026 in the helix H20. Comparison of the results obtained with the crystallographic data on the structure of the central domain of 18S rRNA in the 40S ribosomal subunit led to conclusion that increase of Mg2+ concentrations results in the reorientation of helices H20 and H24 relatively helices H22 and H23 to form a structure, in which these helices are positioned the same way as in 40S subunits. Hence, saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding does, and leads to decreasing of this domain affinity to the protein.

Binding of the human ribosomal protein S13 to the central domain of 18S rRNA

Human ribosomal protein S13 is a structural element of the small subunit of ribosome. It is a homologue of eubacterial ribosomal protein S15, and, besides, it possesses an extended N-terminal region, characteristic of the S15p family in eukaryotes and archaea. In the present study, we investigated binding of recombinant ribosomal protein S13 and its mutants containing deletions or substitutions of amino acid residues in different regions with an RNA transcript corresponding to a fragment of the central domain of 18S rRNA. We found that replacement of ultra-conservative residues H101 and D108 as well as deletions of either 29 C-terminal or 27 N-terminal residues substantially reduced affinity of the protein to the RNA transcript. Deletion of 54 C-terminal or 80 N-terminal residues completely deprived the protein of binding capacity. Using a footprinting assay, we identified sites in the RNA transcript changing their accessibilities to action of hydroxyl radicals under binding of either full-length protein S13 or its mutant lacking 27 N-terminal residues. It is shown that these sites are located mainly in helix H22 of the 18S rRNA and in the region of its junction with helix H20 and are consistent predominantly with contacts of the rRNA with the conserved part of the protein. We concluded that binding of ribosomal protein S13 to 18S rRNA is provided mainly by conserved motifs of the protein corresponding to those motifs in its eubacterial homologue that are involved in the interaction with 16S rRNA in the 30S subunit. Role of the N-terminal region of the protein in its binding to the central domain of 18S rRNA is discussed.

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 5' terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

Protein S20 Binds Two 16S rRNA Sites as Assembly Is Initiated

Ribosomal protein S20 is a primary binding protein that bridges the 5′ domain and the 3′ minor domain of the 16S ribosomal RNA (rRNA) in the 30S ribosomal subunit. Using time-dependent dimethyl sulfate modification, we have determined that as it is bound to 16S rRNA, protein S20 causes rapid protection of bases A246, A274, A279, and A282 in the stem region of helix 11 in the 5′ domain and moderately fast modifications of helix 44 bases A1433 and A1434 in the 3′ minor domain. At a later time, enhancements occur with bases A181and A190 in helix 9, bases A325 and A327 in helix 13, and base C264 at the distal end of helix 11 in the 5′ domain of 16S rRNA. The modifications that occur in the stem region of helix 11 are distant from the binding site of protein S20, as determined from the crystal structure. Simultaneous addition of protein S17 with S20 to the complex significantly alters the modifications caused by protein S20 in the stem region of helix 11 but does not alter the remaining modifications. Our results indicate that protein S20 is binding to at least two alternate 16S rRNA sites during the early assembly process.

Region 1112–1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: Accessibility for complementary interactions and the functional role

The binding of the 18S RNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112-1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5'-untranslated region upstream of the reporter sequence coding for beta-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5' end allowed for the demonstration that the 1112-1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112-1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.

Salt Dependent Folding Energy Landscape of RNA Three-Way Junction

RNAs are highly negatively charged molecules. Salt ions are crucial for RNA folding stability and conformational changes. In the present work, we employ the recently developed tightly bound ion (TBI) model, which accounts for the inter-ion correlations and the fluctuation of ion distributions, to investigate the ion-dependent free energy landscape for the three-way RNA junction in a 16S rRNA domain. The predicted electrostatic free energy landscape suggests that (a) ion-mediated electrostatic interactions cause an ensemble of unfolded conformations narrowly populated around the maximally extended structure and (b) Mg2+ ion-induced correlation effect may help bring the helices to the folded state. Non-electrostatic interactions, such as non-canonical interactions within the junctions and between junctions and helix stems, might further limit the conformational diversity of the unfolded state, resulting in a more ordered unfolded state than the one predicted from the electrostatic effect. Moreover, the folded state is predominantly stabilized by the coaxial stacking force. The TBI-predicted folding stability agrees well with the experimental measurements for the different Na+ and Mg2+ ion concentrations. For Mg2+ solutions, the TBI model, which accounts for the ion correlation effect, gives more accurate predictions than the Poisson-Boltzmann theory, which tends to underestimate the role of Mg2+ in stabilizing the folded structure. Detailed control tests in-dicate that the dominant ion correlation effect comes from the charge-charge Coulombic correlation rather than the excluded volume (size) correlation between the ions. Furthermore, the model gives quantitative predictions for the ion size effect in the folding stability.

Salt-Dependent Folding Energy Landscape of RNA Three-Way Junction

RNAs are highly negatively charged chain molecules. Metal ions play a crucial role in RNA folding stability and conformational changes. In this work, we employ the recently developed tightly bound ion (TBI) model, which accounts for the correlation between ions and the fluctuation of ion distributions, to investigate the ion-dependent free energy landscape for the three-way RNA junction in a 16S rRNA domain. The predicted electrostatic free energy landscape suggests that 1), ion-mediated electrostatic interactions cause an ensemble of unfolded conformations narrowly populated around the maximally extended structure; and 2), Mg 2+ ion-induced correlation effects help bring the helices to the folded state. Nonelectrostatic interactions, such as noncanonical interactions within the junctions and between junctions and helix stems, might further limit the conformational diversity of the unfolded state, resulting in a more ordered unfolded state than the one predicted from the electrostatic effect. Moreover, the folded state is predominantly stabilized by the coaxial stacking force. The TBI-predicted folding stability agrees well with the experimental measurements for the different Na + and Mg 2+ ion concentrations. For Mg 2+ solutions, the TBI model, which accounts for the Mg 2+ ion correlation effect, gives more improved predictions than the Poisson-Boltzmann theory, which tends to underestimate the role of Mg 2+ in stabilizing the folded structure. Detailed control tests indicate that the dominant ion correlation effect comes from the charge-charge Coulombic correlation rather than the size (excluded volume) correlation between the ions. Furthermore, the model gives quantitative predictions for the ion size effect in the folding energy landscape and folding cooperativity.

The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli

Ribosome biogenesis is coupled to the transcription of ribosomal (r)RNAs, which enables an ordered and hierarchical assembly of the ribosome starting with the 5'-terminal domain. We constructed four circular permutants (CPs) of Escherichia coli rRNAs in which the original termini of 16S or 23S rRNAs were genetically connected, and new termini were created elsewhere within the same rRNAs (in helix 33 of 16S rRNA, or in helices 45, 63, and 78 of 23S rRNA). Unexpectedly, all CPs tested were able to rescue E. coli strain Delta7 prrn, which lacks all chromosomal rRNA operons. This result demonstrates that hierarchical assembly from the 5'-terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. However, severe growth defects of all CPs were found in the absence of the DEAD box RNA helicase deaD, indicating that DeaD assists in the efficient assembly of each subunit in the cell.

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE.

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.