Computer modeling 16 S ribosomal RNA

A three-dimensional structure for 16 S RNA has been produced with a computer protocol that is not dependent on human intervention. This protocol improves upon traditional modeling techniques by using distance geometry to fold the molecule in an objective and reproducible fashion. The method is based on the secondary structure of RNA and treats the molecule as a set of double-stranded helices that are linked by flexible single-strands of variable length. Data derived from chemical cross-linking studies of 16 S RNA and tertiary phylogenetic relationships provide the constraints used to fold the molecule into a compact three-dimensional form. Possibly subjective evaluation of the input data are transformed into verifiable quantitative parameters. Relationships based on general locations within the 30 S subunit or on protein-RNA interactions have been specifically excluded. The resolution of the model exceeds that of electron micrographs and approaches that obtained in preliminary X-ray crystal structures. The model size of 245 x 190 x 140 A is compatible with that of the 30 S subunit as determined by electron microscopy. The volume of the model is 1.87 x 10(6) A which is similar to that of the small subunit in a preliminary X-ray crystal structure. The radius of gyration of the model structure of 76 A is intermediate to that seen for partially denatured and fully folded 16 S RNA. Computer graphics are used to display the results in a manner that maximizes the opportunities for human visual interpretation of the models. A format for displaying the structures has been developed that will make it possible for researchers who have not devoted themselves to ribosomal modeling to comprehend and make use of the information that the models embody. On this basis the computer-generated models are compared with models developed by other researchers and with structural data not included in the folding parameter data set.     

Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA

We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.     

Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA

Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.     

An experimentally-derived model for the secondary structure of the 16S ribosomal RNA from Escherichia coli

Ribonucleoprotein fragments are isolated by mild ribonuclease digestion of E. coli 30S ribosomal subunits, and are deproteinized and subjected to a second partial digestion. Base-pairing between the resulting small RNA fragments is investigated using the two-dimensional gel electrophoresis procedure already reported (see Ref. 1). The interactions thus found are incorporated into a secondary structure model covering approximately 80% of the 16S RNA.     

Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

Probing the stabilizing effects of modified nucleotides in the bacterial decoding region of 16S ribosomal RNA

1. Download : Download full-size image

     

Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli

The complete range of published sequences for ribosomal RNA (or rDNA), totalling well over 50,000 bases, has been used to derive refined models for the secondary structures of both 16S and 23S RNA from E. coli. Particular attention has been paid to resolving the differences between the various published secondary structures for these molecules. The structures are described in terms of 133 helical regions (45 for 16S RNA and 88 for 23S RNA). Of these, approximately 20 are still tentative or unconfirmed. A further 20 represent helical regions which definitely exist, but where the detailed base-pairing is still open to discussion. Over 90 of the helical regions are however now precisely established, at least to within one or two base pairs.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Probing the assembly of the 3' major domain of 16 S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19

We have studied the effect of assembly of ribosomal proteins S7, S9 and S19 on the accessibility and conformation of nucleotides in 16 S ribosomal RNA. Complexes formed between 16 S rRNA and S7, S7 + S9, S7 + S19 or S7 + S9 + S19 were subjected to a combination of chemical and enzymatic probes, whose sites of attack in 16 S rRNA were identified by primer extension. The results of this study show that: (1) Protein S7 affects the reactivity of an extensive region in the lower half of the 3' major domain. Inclusion of proteins S9 or S19 with S7 has generally little additional effect on S7-specific protection of the RNA. Clusters of nucleotides that are protected by protein S7 are localized in the 935-945 region, the 950/1230 stem, the 1250/1285 internal loop, and the 1350/1370 stem. (2) Addition of protein S9 in the presence of S7 causes several additional effects principally in two structurally distal regions. We observe strong S9-dependent protection of positions 1278 to 1283, and of several positions in the 1125/1145 internal loop. These findings suggest that interaction of protein S9 with 16 S rRNA results in a structure in which the 1125/1145 and 1280 regions are proximal to each other. (3) Most of the strong S19-dependent effects are clustered in the 950-1050 and 1210-1230 regions, which are joined by base-pairing in the 16 S rRNA secondary structure. The highly conserved 960-975 stemp-loop, which has been implicated in tRNA binding, appears to be destabilized in the presence of S19. (4) Protein S7 causes enhanced reactivity at several sites that become protected upon addition of S9 or S19. This suggests that S7-induced conformational changes in 16 S rRNA play a role in the co-operativity of assembly of the 3' major domain.     

16S ribosomal RNA analysis of Filibacter limicola indicates a close relationship to the genus Bacillus

The phylogenetic relationship of the Gram-negative, filamentous gliding bacterium Filibacter limicola was analysed by 16S rRNA oligonucleotide cataloguing. In contrast to the proposed membership of this asporogenous species in the Flexibacteriaceae, Filibacter limicola clusters phylogenetically with the Gram-positive eubacteria Bacillus pasteurii, Sporosarcina ureae and the asporogenous species Planococcus citreus. The genetic relationship is supported by several common phenotypic properties.     

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.     

Sequence studies on 16S ribosomal RNA from a blue-green alga

Summary The 16S ribosomal RNA of the blue green algaAnacystis nidulans has been characterized in terms of the oligomers generated by digestion with T1 ribonuclease.A. nidulans by this criterion is definitely a procaryote; being no more distant from Bacilli or Enterics than the latter two are from one another.A. nidulans appears to be somewhat more closely related to the Bacilli than to the Enterics.This is contribution III in a series entitled Procaryote phylogeny.     

Precursors of 5 S ribosomal RNA in Bacillus subtilis

Bacillus subtilis 168 accumulates subnormal quantities of mature 5 S ribo-somal RNA in the presence of inhibitors of protein synthesis, such as chloramphenicol, or during pulse-labeling experiments. However, two RNA species, evidently precursors of m5 rRNA and therefore designated as p5_A and p5_B, do accumulate under these conditions. These RNA species are substantially longer than B. subtilis m5 rRNA: p5_A is about 179 nucleotides in length and p5_B is composed of approximately 152 nucleotides. The sum of p5_A, p5_B and m5 rRNA accumulating in the absence of protein synthesis, less excess chain length associated with p5_A and p5_B, equals the expected quantities of m5 rRNA in growing cells. p5_A and p5P_B both contain all t₁ RNase-generated oligonucleotides characteristic of m5 rRNA plus additional sequences. At least the 5′ termini of p5_A and p5_B differ from that of m5. If chloramphenicol is removed from a culture in which p5_A and p5_B have accumulated and further RNA synthesis is inhibited, then a quantitative reciprocal loss of p5_A and p5_B occurs as m5 rRNA accumulates. No evidence suggests any p5_A to p5_B transition under these conditions.