Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains (original) (raw)
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Journal of Molecular Biology, 2000
The Escherichia coli 23 S and 5 S rRNA molecules have been ®tted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an un®ltered version of the recently published 50 S reconstruction at 7.5 A Ê resolution. At this resolution, the EM density shows a well-de®ned network of ®ne structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A Ê or 20 A Ê . Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modi®ed bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.
Nucleic Acids Research, 1988
We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein SI5 using a variety of enzymatic and chemical probes. RNase Ti and nuclease S 1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(i-methylmorpholino)-ethyl)-carbodiimide-p-toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule;
Molecular Biology, 2001
Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coli and Thermus thermophilus S7 with a fragment of the 3' domain of the E. coli 16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA-protein interactions.
An assembly landscape for the 30S ribosomal subunit
Nature, 2005
Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions.
eLife, 2014
Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3' domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3'-domain is unanchored and the 5'-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.
Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure
Nucleic Acids Research, 1981
Extensive studies in our laboratory using different ribonucleases ressulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies gave precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coZi 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.
Analysis of the Ribosome Large Subunit Assembly and 23 S rRNA Stability in Vivo
Journal of Molecular Biology, 1996
The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunitin vivowas studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of anE. coli23 S rRNA gene. The mutant genes were expressedin vivousing an inducibletacpromoter. Mutant species of 23 S rRNA, containing deletions between
European Journal of Biochemistry, 1973
1. 30-S ribosomal subparticles from Escherichia mli were hydrolysed with ribonuclease TI, pancreatic ribonuclease or micrococcal nuclease in the presence of 2 M urea, and various concentrations of magnesium and ethanol. The RNAprotein fragments produced were separated on 50l0 polyacrylamidelagarose composite gels, and fractions from these gels were subjected to protein analysis on i7.5O/, periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published. 2. A wide range of RNA * protein fragments was obtained by this procedure, each containing a few specific ribosomal proteins. The strict criteria already published for determining the specificity of the proteins in each fragment were applied. The RNAprotein fragments divide into two distinct groups, those containing some or all of proteins 57, S9, SiO, 513, Si4 and Si9, and those containing some or all of proteins S4, 55, S6, S8, Sii, Sl5, S16(i7), 518 and S20. Proteins S1, S2, 53, Si2 and 521 were not found in specific fragments. 3. The individual proteins found together in specific RNAprotein fragments are interpreted as being close neighbours in the 30-S particle. The range of fragments observed is sufficient to enable the data to be combined with Nomura's "assembly map)' and data from protein crosslinking experiments, into a preliminary three-dimensional arrangement of the proteins. I n previous papers we have described a method for the analysis of ribonucleoprotein (RNAprotein) fragments from Escherichia coli ribosomes [i], and have used this method to characterize a series of specific fragments from the 30-5 particle [2]. I n this paper we present a further series of fragments, obtained by mild nuclease digestion of the 30-S ribosome with ribonuclease TI, micrococcal nuclease or pancreatic ribonuclease. These two series of fragments account for 16 out of the 21 ribosomal proteins, and the data have been combined with the assembly map of Nashimoto et d. [3], into a preliminary threedimensional model. The protein arrangement takes account of results obtained by cross-linking of ribosomal proteins with bi-functional reagents [4,5,6], and a possible linear sequence of some of the proteins along the ribosomal RNA is also discussed. Abbreviations. RXA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine. Enzymee. Ribonuclease TI (.EC 2.7.7.26) ; pancreatic ribonuclease (EC 2.7.7.16) ; micrococcal nuclease (EC 3.1.4.7). Definition. Azao unit is the quantity of material contained in I ml of a solution which has an absorbance of I at 260 nxn, when measured in a 1-cm path-length cell. MATERIALS AND METHODS Preparation of Ribosomes Radioactive and non-radioactive 30-5 ribosomal sub-particles from E. coli MRE 600 (obtained from MRE, Porton, U.K.) were prepared exactly as described previously [2], except that the isolated subparticles were kept stored at-20 "C in 10 mM Tris-HC1 pH 7.8, 0.3 mM magnesium acetate, Containing 10-20°/0 ethanol. The ethanol was only dialysed away immediately before use in hydrolysis reactions. Separation and Analysis of RNA .Protein Fragments Radioactive 30-S ribosomes were hydrolysed with ribonuclease TI, pancreatic ribonuclease, or micrococcal nuclease (all from Sigma) for 4.5 h at room temperature. Reaction mixtures contained 8-10 ABso units of ribosomes in 0.2-0.4 ml. The hydrolysates were separated and analysed by electrophoresis on 501, polyacrylamide/0.5 agarose composite gel slabs [7], exactly as before [2]. The hydrolysis buffers contained i0 mM Tris-HC1 pH 7.8, 20 mM KCI, and 2 M urea [2], with various amounts of added magnesium acetate, ethanol and enzyme, as