Preparation of ribosomes

The ribosome provides an easily accessible source of endogenous RNA-protein complexes that participate in the overall process of translation. In this section, we present protocols for obtaining salt-washed ribosomes from E. coli and mammalian cell-lines. However, if highly active vacant couples devoid of tRNAs and mRNAs is the aim, it is necessary to use more elaborate protocols that include dissociation of bacterial 70 S tight couples into subunits,17 

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Although there may be some exceptions, it appears likely that the majority of the proteins in muscle fibers are synthesized in situ. Preparations of ribosomes can be made from muscle which are capable of synthesizing protein under conditions similar to those used for other tissues (Yl). Varying estimates have been made of the rate of turnover of muscle proteins (PIO) older estimates of protein half-lives are probably too long owing to error resulting from the reutilization of the labeled... 

CpCpApCpCpA and this hexanucleotide has been prepared from appropriately protected trinucleotides using a sulphonyl chloride as condensing agent.2 (3 )-0-Glycyl esters of Cpl and dCpA have been prepared as potential substrates of ribosomal peptidyl transferase. While the glycyl ester of Cpl released the polypeptide chain from polylysyl-tRNA in a ribosomal system from E. coli, the dCpA derivative showed little activity. 

Preparation of rough microsomes and ribosome-free reticulocyte lysate... 

Of particular interest, the data depicted in Fig. 4.1 demonstrate that ribosomes remain membrane-bound after termination and, surprisingly, are recovered predominantly as 80S rather than the predicted posttermination 40S + 60S subunits. It would be of high interest to determine if 80S post-termination, ER-bound ribosomes are competent for initiation, to identify the cohort of initiation factors necessary for initiation with 80S ribosomes, and also to determine whether initiation and scanning require dissociation of the 80S couplet. Methods to address these questions are under development but unavailable at the time of preparation of this contribution. 

The in vitro transcribed RNAs are phenol-chloroform extracted, ethanol-precipitated, and washed once with 70% ethanol. The RNA pellet is resuspended in 30 1 H20 and loaded to DyeEx 2.0 spin columns (Qiagen) to remove free nucleotides the RNA is then quantified by spectrophotometry. Approximately 20 to 30 pg of RNA is obtained from one reaction. For the initial preparation of transcripts, we would examine the quality and quantity of synthesized RNA by separating them on 1% denaturing agarose gels with total cell RNA preparation as a size maker (28S and 18S ribosomal RNAs... 

Coupling of affinity molecules to surfaces also can be enhanced by the use of discrete PEG linkers. Nishimura et al. (2005) modified an amino surface with a NHS-PEG -maleimide crosslinker to create a hydrophilic self-assembled monolayer (SAM) surface that was thiol reactive for the conjugation of sulfhydryl-modified RNAs. This array then was used to investigate the binding specificity of synthetic kanamycins with selected RNA sequences to prove the specific interaction of ribosomal RNA with this molecule. The PEG linkers on surfaces provide lower nonspecific binding character than alkyl linkers, when preparing SAM surfaces for affinity interactions. 

As discussed above, it appears from physical studies, especially with the NMR technique, that the tertiary structure of ribosomal proteins isolated in the presence of 6 M urea and then carefully renatured under appropriate conditions is very similar to those proteins prepared in the complete absence of urea. 

For a number of years now we have been involved in crystallization of bacterial ribosomal particles. From the very beginning of our studies, the crucial need for a stable, very intense, and perfectly focussed synchrotron beam was evident, even for preliminary and basic information (e.g. whether crystals diffract at all). Thus our studies have always been dependent on the availability of synchrotron beam time and hampered by only partial (and very occasional) feedback to assess our experimental procedures for growing bacteria, preparing the ribosomes and obtaining crystals. 

Termination of translation involves release of the completed protein and preparation of the ribosomal subunits for another cycle. The entire process is powered by the hydrolysis of ATP and GTP. The ATP is utilized in a three-step process for the activation of the amino acids, which become linked to the appropriate tRNAs (Eqs. 17-36 and 29-l).61a If the inorganic pyrophosphate that is formed is hydrolyzed, two molecules of ATP are required for activation of each amino acid molecule. In addition, at least two molecules of GTP are hydrolyzed to GDP and inorganic phosphate within the ribosome for each peptide linkage formed. 

Many specific parts of ribosomal RNA molecules and specific proteins within the intact ribosome were located prior to the determination of high resolution crystal structures. One major approach was the use of immunoelectron microscopy. Antibodies to specific ribosomal proteins or to special sites in the RNA were prepared, and electron microscopy was used to map the binding sites of the antibodies on the ribosomal... 

Within bacterial cells the assembly of ribosomes is coupled to rRNA synthesis and requires only 1-2 minutes.115 In the laboratory both the 30S ribosomal subunits116 and the 50S subunits117-121 of E. coli can be completely dissociated into individual protein and RNA molecules and can be reconstituted in a functional form. This is true for both natural 16S or 23S RNA or for RNAs prepared by in vitro transcription. In these reassembly experiments, which were pioneered by Nomura,116 it was found that the order of addition of the protein is important. Some proteins bind... 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

The cell-free system prepared from washed embryos has much higher translational activity than the conventional system (compare Fig. 3A and B). When 5 -capped dihydrofolate reductase (DHFR) mRNA containing 549 nt of 3 UTR with a pA tail was incubated with newly prepared as well as conventional extract, there was almost linear kinetics in DHFR synthesis over 4 h, compared with the regular system, which ceased to function after 1.5 h. Further, when washed extract in the reaction volume was increased to 48%, amino acid incorporation occurred initially at a rate twice that of 24% extract, and then stopped after 1 h. However, this pause was caused by a shortage of substrates rather than an irreversible inactivation of ribosomes or factors necessary for translation addition of amino acids, ATP, and GTP after cessation of the reaction (arrow) restarted... 

Prepare a 2-pg/mL solution of ribosomal RNA standard provided in TE. Prepare a series of standard RNA concentrations from 0 to 2 pg/mL. Mix equal volumes (100 pL) of diluted RiboGreen Reagent and standard RNAs. Measure the sample fluorescence using a TD-700 fluorometer (excitation approx 480 nm, emission approx 520 nm) and prepare the standard curve. 

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