Ribosomes of E. coli

Once the amino acid has been bound to its tRNA, it can pass to the next phase of protein synthesis, involving its interaction with mRNA, which takes place on the ribosome, a molecular machine of enormous complexity. The ribosome of E. coli is a ribonucleoprotein assembly of molecular weight 2700 kDa, and sedimentation constant of 70S9. It is made up of roughly two-thirds RNA and one-third protein, and can be separated into a small (30S) and a large (50S) subunit. The 30S subunit contains 21 proteins and one 16S RNA molecule, while the large subunit has 34 different proteins and two RNA molecules, one 23S and one 5S. Despite its size and complexity, the structure of both ribosomal subunits has been determined to atomic resolution (Figure 4.32), and very recently the atomic structure of the 70S ribosome has been determined at 2.8 A resolution (Selmer et al., 2006). 

List the kinds and numbers of macromolecular components of the prokaryotic ribosome. Give the mass, sedimentation coefficient, and dimensions of the ribosome of E. coli. 

Ammonium aurintricarboxylate (LXIII) along with several other triphenyl-methane dyes has been found active in cell-free systems at 10 m in preventing the binding of a bacteriophage RNA to the ribosomes of E. coli. In viral... 

Ribosomes are involved in protein biosynthesis, where they mediate the interaction between mRNA and aminoacylated tRNA in order to lead to polypeptide chain formation. The 70S ribosomes of E. coli are composed of two subunits, the SOS and 30S subunits. The SOS subunit (A/ 1,4S0,000 68% RNA) is constructed from SS and 23S rRNA and 32 different single proteins designated LI to L34 (except that there are 4 copies of L7 and L12), while the 30S subunit 8S0,000 S9% RNA) is formed from 16S rRNA and 21 different proteins designated SI to S21. The values are derived from the full primary sequence, so do not include contributions from Mg, and spermidine binding. The three applications of solution scattering to the ribosome [24-26,77] can be classified as (a) study of the 70S, SOS and 30S ribosomes (b) study of the free proteins and RNAs and some complexes that can be formed between them (c) determination of the quaternary arrangement of the proteins within the 30S and SOS subunits by triangulation. These are discussed in turn [4S,48,424-446],... 

Differential inhibitory effects of antibiotics on the synthesis of membrane and cytoplasmic proteins may be due to differences in the ribosomes themselves. Differences in the ribosomes of E, coli may result from the protein components or the RNA components. In fact, some ribo-somal proteins have been shown to be present in only a fraction of ribosomes, resulting in a heterogeneity of ribosomes. The fact that there are several nonlinked ribosomal RNA genes in an E. coli genome may reflect the differentiation of ribosomes. This differentiation of ribosomes may be the direct cause of the different sensitivities of ribosomes to antibiotics. Alternatively, the different sensitivities of ribosomes to antibiotics could be caused indirectly as a result of compartmentalization of ribosomes inside the cell. Existence of a subpopulation of ribosomes with different sensitivities has been suggested for MS2 phage protein synthesis and for a and 0 chains of hemoglobin in rabbit erythrocytes. ... 

Pearson and coworkers investigated several antibiotic-resistant strains of E. coli in an elegant and thorough study. Using a combination of MALDI-TOF and electrospray LC-MS they found that varying subtle mutations in ribosomal proteins occur in different strains that are resistant to various anti-... 

More recently, however, this mechanism was questioned by several researchers [156-158]. In the case of E. coli 50S subunits, the pH-dependent modification of A2451 by DMS (A2486 in Haloarcula marismortui) occurred only in its inactive conformation [157]. Under the conditions wherein the ribosomes were fully active, in contrast, the modification of A2451 by DMS could not be recognized. These results indicate that A2451 becomes shielded from chemical modification by a conformational change upon activation. Thus, it was concluded that the conditional modification of A2451 by DMS... 

Our first microcrystals were of Bacillus stearothermophilus SOS subunits and of 70S ribosomes from E. coli they were mainly grown from lower alcohols, toluene, or chloroform. Each ribosomal preparation required slightly different crystallization conditions, and often the preparation had almost been exhausted by the time conditions were optimised. We also found that crystals grew from active particles only. 

Karimi R, Ehrenberg M (1994) Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes. Eur J Biochem 226 355-360 Karimi R, Ehrenberg M (1996) Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes. EMBOJ 15 1149-1154... 

All ribosomes have two subunits, and each subunit contains several protein chains and one or more chains of RNA (ribosomal RNA, or rRNA). In the ribosome from E. coli, the smaller of the two subunits is known as the 30S subunit and the larger is referred to as the 50S subunit. (The unit S stands for Svedberg, a measure of how rapidly a particle sediments in a centrifuge.) The two subunits combine to form the active 70S ribosomal assembly. The special RNA molecules that are a part of the ribosome are quite distinct from messenger or transfer RNA molecules, and they play important roles in forming the overall ribosomal quaternary structure and in aligning mRNA and tRNA molecules during protein biosynthesis. 

Using a tubular-bowl centrifuge, calculate the sedimentation velocity of a 70S ribosome (from E. coli, diameter 0.02 pm) in water at 20 °C. The rotational speed and distance from the center are 30 000 rpm and 10 cm, respectively. 

The cytoplasm of E. coli contains about 15,000 ribosomes, thousands of copies each of about 1,000 different enzymes, numerous metabolites and cofactors, and a variety of inorganic ions. The nucleoid contains a single, circular molecule of DNA, and the cytoplasm (like that of most bacteria) contains one or more smaller, circular segments of DNA called plasmids. In nature, some plasmids confer resistance to toxins and antibiotics in the environment. In the laboratory, these DNA segments are especially amenable to experimental manipulation and are extremely useful to molecular geneticists. 

Compare the following the diameters of (a) a carbon atom in an organic molecule (b) a bacterial cell, e.g. of E. coli (c) a human red blood cell (d) a ribosome (e) the length of a peptide unit in an extended polypeptide chain (f) the length of the carbon atom chain in an 18-carbon fatty acid. 

Figure 5-32 (A) A three-dimensional computer graphics model proposed by Brimacombe et a/.3 11 for the single chain of E. coli 16S ribosomal RNA. The helices are depicted as cylinders, which are all connected. The small dark squares denote the positions of artificially formed RNA-protein crosslinks, marked with the appropriate protein number. For proteins exhibiting more than one crosslink site (e.g., SI7), the sites are denoted A or B, in each case A being the site nearer to the 5 terminus of the 16S RNA. (B) Stereoscopic view of tentative atomic model of 16S RNA in the 30S ribosomal subunit. The viewing direction is different from that in (A). From Mueller and Brimacombe.342 Courtesy of Richard Brimacombe.

In 1961, Jacob and Monod postulated messenger RNA (mRNA) as a short-lived polynucleotide.30 32 33 An abundance of additional evidence supported the proposal. For example, RNA molecules produced after infection of E. coli by bacteriophage T4 underwent hybridization (Chapter 5) with denatured DNA of the bacteriophage. Furthermore, this virus-specific mRNA became associated with preexisting bacterial ribosomes and provided the template for synthesis of phage proteins.34 The experiment provided direct evidence for transcription of mRNA from genes of the viral DNA. 

The many thousands of proteins present in each cell are made within the ribosomes, which are able to read each specific mRNA that comes their way. While ribosomes appear as little more than blurred dots in most electron micrographs, the 15,000 ribosomes of one cell of E. coli represent one-fourth of the total mass of the cell. Eukaryotic cells contain many times more of these little molecular machines. When ribosomes were first observed in the early 1950s,1 3 nobody could imagine either their composition or their function. 

The sequences of all three pieces of RNA in the E. coli ribosomes are known as are those from many other species. These include eukaryotic mitochondrial, plas-tid, and cytosolic rRNA. From the sequences alone, it was clear that these long molecules could fold into a complex series of hairpin loops resembling those in tRNA. For example, the 16S rRNA of E. coli can fold as in Fig. 29-2A and eukaryotic 18S RNA in a similar way (Fig. 29-4).38/39/67 69 The actual secondary structures of 16S and 18S RNAs, within the folded molecules revealed by X-ray crystallography, are very similar to that shown in Fig. 29-2A. Ribosomal RNAs undergo many posttranscriptional alterations. Methylation of 2 -hydroxyls and of the nucleic acid bases as well as conversion to pseudouridines (pp. 1638-1641) predominate over 200 modifications, principally in functionally important locations that have been found in human rRNA.69a... 

Figure 29-4 Structure of 23S-28S ribosomal RNAs. (A) The three-dimensional structure of RNA from the 50S subunit of ribosomes of Halocirculci marismortui. Both the 5S RNA and the six structural domains of the 23S RNA are labeled. Also shown is the backbone structure of protein LI. From Ban et al.17 Courtesy of Thomas A. Steitz. (B) The corresponding structure of the 23S RNA from Thermus thermophilus. Courtesy of Yusupov et al.33a (C) Simplified drawing of the secondary structure of E. coli 23S RNA showing the six domains. The peptidyltransferase loop (see also Fig. 29-14) is labeled. This diagram is customarily presented in two halves, which are here connected by dashed lines. Stem-loop 1, which contains both residues 1 and 2000, is often shown in both halves but here only once. From Merryman et al.78 Similar diagrams for Haloarcula marismortui17 and for the mouse79 reveal a largely conserved structure with nearly identical active sites. (D) Cryo-electron microscopic (Cryo-EM) reconstruction of a 50S subunit of a modified E. coli ribosome. The RNA has been modified genetically to have an...

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