A Map of Protein-rRNA Distribution in the 70 SEscherichia coli Ribosome

Abstract

Neutron scattering exploits the enormous scattering difference between protons and deuterons. A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations. This extensive data set is analyzed using a novel method. The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y. H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R. K., and Agrawal, R. K. (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm. Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves. Twelve independent reconstructions starting from random approximations yielded reproducible results. The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA. 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods. The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps.Ribosomes are supramolecular complexes responsible for protein synthesis in all organisms. Each of the two unequal ribosomal subunits is a complicated assembly of proteins and nucleic acids. Thus, the prokaryotic 70 S ribosome from Escherichia coli has a total molecular mass of about 2.3 ϫ 10 6 Da and consists of a 30 S and 50 S subunit (1). The 30 S subunit contains 21 individual proteins and a single 16 S rRNA molecule, and the 50 S subunit, 33 proteins and two rRNA molecules (5 S rRNA ϩ 23 S rRNA). In both subunits the rRNA moieties account for about two-thirds of the mass. A better description of the interactions between proteins and rRNA in the ribosome is of paramount importance for the understanding of the structural mechanism of protein synthesis.The structure of the ribosome has been extensively studied for decades using various methods. In the last years, cryoelectron microscopy (cryo-EM) 1 yielded models of the overall shape of the ribosome at a resolution of 1.5 nm (2-5). Recent tremendous progress in the x-ray crystallography provided electron density maps down to 0.78 nm for ribosomal complexes containing tRNAs (6) and to 0.45 nm for the individual ribosomal subunits (7-10). Despite these remarkable achievements, limited information has so far been obtained about the spatial distribution of rRNA and proteins in the ribosome. Using the x-ray maps, six ribosomal proteins were positioned in the small (7, 10) and four in the large subunit (8). The low contrast between ribosomal proteins and rRNA for both electrons and x-rays makes it d...

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

“…The in-solution folded C-terminus conformation of hRNF4 is consistent with previous studies indicating that this conformation is required for RNF4 activity [31]. We used the SAXS-based method MONSA that employs a multiphase modeling to fit simultaneously multiple curves, and thus applies a contrast method to define interaction of protein and DNA constituents within the larger complex [33,34]. We collected SAXS data on apo-hRNF4 ( Supplementary Fig S3), hRNF4 bound to a 12-bp double-stranded (ds) DNA oligonucleotide, and the 12-bp oligonucleotide alone ( Supplementary Fig S4).…”

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

“…The in-solution folded C-terminus conformation of hRNF4 is consistent with previous studies indicating that this conformation is required for RNF4 activity [31]. We used the SAXS-based method MONSA that employs a multiphase modeling to fit simultaneously multiple curves, and thus applies a contrast method to define interaction of protein and DNA constituents within the larger complex [33,34]. We collected SAXS data on apo-hRNF4 ( Supplementary Fig S3), hRNF4 bound to a 12-bp double-stranded (ds) DNA oligonucleotide, and the 12-bp oligonucleotide alone ( Supplementary Fig S4).…”

A picornaviral loop-to-loop replication complex

“…imentally determined molecular weights for 3C pro and 3C pro -SLD, as well as a lysozyme standard (calculated from measured I(0) values with equation (1)) agree well with the expected values based on amino acid and nucleotide sequences. The experimental R g value for 3C pro derived from its P(r) agrees with the theoretical value from the NMR structure (16.7 Å, calculated using the program CRY-SOL (Svergun and Nierhaus, 2000)). In contrast, the SLD molecular weight determined from I(0) was approximately twice the expected value indicating that under our experimental conditions SLD is not a monomer in solution.…”

Small-angle scattering studies of biological macromolecules in solution

“…Each sphere is assigned either to solvent, to protein or to ribosomal RNA (rRNA) moieties to simultaneously fit all the scattering curves. The resulting 3 nm resolution model represents the volumes occupied by the rRNA and protein moieties in the entire ribosome [165]. The predicted protein-rRNA map is in remarkably good agreement with the later high-resolution crystallographic models of the ribosomal subunits from other species [88,140,166].…”

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

“…The in-solution folded C-terminus conformation of hRNF4 is consistent with previous studies indicating that this conformation is required for RNF4 activity [31]. We used the SAXS-based method MONSA that employs a multiphase modeling to fit simultaneously multiple curves, and thus applies a contrast method to define interaction of protein and DNA constituents within the larger complex [33,34]. We collected SAXS data on apo-hRNF4 ( Supplementary Fig S3), hRNF4 bound to a 12-bp double-stranded (ds) DNA oligonucleotide, and the 12-bp oligonucleotide alone ( Supplementary Fig S4).…”

A picornaviral loop-to-loop replication complex

“…imentally determined molecular weights for 3C pro and 3C pro -SLD, as well as a lysozyme standard (calculated from measured I(0) values with equation (1)) agree well with the expected values based on amino acid and nucleotide sequences. The experimental R g value for 3C pro derived from its P(r) agrees with the theoretical value from the NMR structure (16.7 Å, calculated using the program CRY-SOL (Svergun and Nierhaus, 2000)). In contrast, the SLD molecular weight determined from I(0) was approximately twice the expected value indicating that under our experimental conditions SLD is not a monomer in solution.…”

Small-angle scattering studies of biological macromolecules in solution

“…Each sphere is assigned either to solvent, to protein or to ribosomal RNA (rRNA) moieties to simultaneously fit all the scattering curves. The resulting 3 nm resolution model represents the volumes occupied by the rRNA and protein moieties in the entire ribosome [165]. The predicted protein-rRNA map is in remarkably good agreement with the later high-resolution crystallographic models of the ribosomal subunits from other species [88,140,166].…”

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

“…The in-solution folded C-terminus conformation of hRNF4 is consistent with previous studies indicating that this conformation is required for RNF4 activity [31]. We used the SAXS-based method MONSA that employs a multiphase modeling to fit simultaneously multiple curves, and thus applies a contrast method to define interaction of protein and DNA constituents within the larger complex [33,34]. We collected SAXS data on apo-hRNF4 ( Supplementary Fig S3), hRNF4 bound to a 12-bp double-stranded (ds) DNA oligonucleotide, and the 12-bp oligonucleotide alone ( Supplementary Fig S4).…”

A picornaviral loop-to-loop replication complex

“…imentally determined molecular weights for 3C pro and 3C pro -SLD, as well as a lysozyme standard (calculated from measured I(0) values with equation (1)) agree well with the expected values based on amino acid and nucleotide sequences. The experimental R g value for 3C pro derived from its P(r) agrees with the theoretical value from the NMR structure (16.7 Å, calculated using the program CRY-SOL (Svergun and Nierhaus, 2000)). In contrast, the SLD molecular weight determined from I(0) was approximately twice the expected value indicating that under our experimental conditions SLD is not a monomer in solution.…”

Small-angle scattering studies of biological macromolecules in solution

“…Each sphere is assigned either to solvent, to protein or to ribosomal RNA (rRNA) moieties to simultaneously fit all the scattering curves. The resulting 3 nm resolution model represents the volumes occupied by the rRNA and protein moieties in the entire ribosome [165]. The predicted protein-rRNA map is in remarkably good agreement with the later high-resolution crystallographic models of the ribosomal subunits from other species [88,140,166].…”