A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a threedimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chlororibosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.chloroplast protein synthesis ͉ cryo-EM structure ͉ ribosomal RNA ͉ plastid ribosome recycling factor ͉ protein synthesis C ellular organelles, like mitochondria and chloroplasts, possess their own genome and components of gene-expression system, including ribosomes (for reviews, see refs. 1 and 2). The chloroplast (or plastid) ribosome (henceforth referred to as the ''chlororibosome'') is specialized in that it is involved in the biosynthesis of only the small number of proteins encoded by the chloroplast genome. The complete genome sequence of the spinach (Spinacea oleracea) chloroplast identified a total of 146 genes, of which 98 encode protein products, and 45 represent structural RNAs (3). The majority of the chloroplast-encoded proteins is targeted to the thylakoid membranes and include components of the ATP synthase, cytochrome b/f, and especially photosystem I and II complexes (3). In addition, chloro-ribosomes translate NADH dehydrogenase, the large subunit of RuBisCO, RNA polymerase subunits, and a distinct subset of ribosomal proteins: 12 from the small (30S) subunit and 8 from the large (50S) subunit, and the rest are nuclear-encoded and imported into the chloroplast.The origin of chloroplasts is understood to be through an early endosymbiotic event between a photosynthetic prokaryotic ancestor related to cyanobacteria and a eukaryotic cell (4). In fa...

show abstract

“…We have computationally separated the RNA and protein components (12) within both subunits of the chloro-ribosome (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Sharma

Wilson

Datta

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

146

View full text Add to dashboard Cite

show abstract

“…We made use of the separation of the 11.5-Å cryo-EM map into a ''protein only'' and an ''RNA only'' map, achieved on the basis of histogram information and region growing (see Materials and Methods and ref. 31). In the protein-only map that we obtained by using this method, protein masses occur in clusters.…”

Section: Resultsmentioning

confidence: 99%

“…Another difference map was calculated between the protein-only portion of the cryo-EM map of the 30S subunit (isolated in this lab by differentiating proteins from nucleic acids based on density histogram information and a technique of region growing; see ref. 31), and the fitted, filtered protein portion of the x-ray map (25). To determine the position of the mRNA, a third difference map was computed by subtracting cryo-EM maps of 70S ribosomes with and without mRNA (see ref.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA

Sengupta

Agrawal

Frank

2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

149

115

View full text Add to dashboard Cite

S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. Thus far, S1 has eluded structural determination. We have identified the S1 protein mass in the cryo-electron microscopic map of the Escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1. According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data. Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion. The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit. Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine-Dalgarno sequence, explains the protein's role in the recognition of the 5 region of mRNA. P rotein S1 is the largest ribosomal protein, 68 kDa (1), present in the small subunit of the Escherichia coli 70S ribosome. It has been implicated in the selection of the translation start site on the 30S subunit (2). It has been suggested that a pyrimidinerich region upstream of the Shine-Dalgarno (SD) sequence of mRNA interacts with protein S1 and serves as one of the ribosome recognition sites (3). Protein S1 has been reported to be necessary in some cases for translation initiation (4) and for translation elongation (5). It is the only ribosomal protein that has a high affinity for mRNA (6). As a ribosomal protein, S1 is strikingly atypical. Association of S1 to the ribosome is weak and reversible (ribosome preparations often contain less than stoichiometric amounts of protein S1; ref. 7), whereas most other ribosomal proteins are strongly bound. Furthermore, S1 is an acidic protein, whereas all other ribosomal proteins, with the exception of protein L7͞L12, are basic.The S1 protein consists of 557 amino acid residues. Mild trypsin digestion cleaves S1 into two fragments (8), whereas cleavage with cyanogen bromide yields three different fragments (9). The N-terminal fragment from the latter cleavage contains amino acids 1-193, the middle fragment contains amino acids 224-309, and the C-terminal fragment contains amino acids 332-547 (9). The N-terminal domain binds to the platform region of the 30S subunit (10), whereas the C-terminal domain has been suggested to interact with RNAs (11). S1 has been reported to have an elongated shape in solution (12, 13). Neutron scattering (11) and fluorescence anisotropy experiments (14) indicate that the protein maintains its elongated shape even in its ribosome-bound state.So far, no x-ray crystallographic study has been reported for S1. However, an NMR structure of a 75-aa fragment of S1-type polynucleotide phosphorylase of E. coli, which corresponds to the C-terminal domain of S1, has been determined (15). The structure consists of a five-stranded antiparallel ␤-barrel, a structural motif known...

show abstract

“…Separation of cryo-EM density into its two components has nevertheless been achieved, (54) on the basis of information that became available once known proteins and RNA elements could be placed with confidence in low-resolution X-ray maps. (55,56) Density histogram information from the equivalent places in the cryo-EM map of the E. coli ribosome establishes the signatures of``RNA-only'' and``protein-only'' regions ( Fig.…”

Section: Protein±rna Separationmentioning

confidence: 99%

Cryo‐electron microscopy as an investigative tool: the ribosome as an example

Frank

2001

BioEssays

Self Cite

View full text Add to dashboard Cite

Cryo-electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three-dimensional reconstruction, cryo-EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA-protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X-ray structures of ribosomal subunits have become available, conformational changes observed by cryo-EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X-ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery.

show abstract

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

Cited by 37 publications

References 72 publications

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA

Cryo‐electron microscopy as an investigative tool: the ribosome as an example

Contact Info

Product

Resources

About