Eukaryotic ribosomes are larger. They consist of a 60S large subunit and a 40S small subunit, which come together to form an 80S particle having a mass of kd, compared with kd for the prokaryotic 70S ribosome. Initiator tRNA. In eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine. However, as in prokaryotes, a special tRNA participates in initiation.
The initiating codon in eukaryotes is always AUG. This scanning process in eukaryotic protein synthesis is powered by helicases that hydrolyze ATP. In almost all cases, eukaryotic mRNA has only one start site and hence is the template for a single protein.
In contrast, a prokaryotic mRNA can have multiple Shine-Dalgarno sequences and, hence, start sites, and it can serve as a template for the synthesis of several proteins. Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate.
The prefix eIF denotes a eukaryotic initiation factor. For example, eIF-4E is a protein that binds directly to the 7-methylguanosine cap Section The difference in initiation mechanism between prokaryotes and eukaryotes is, in part, a consequence of the difference in RNA processing. In contrast, pre-mRNA must be processed and transported to the cytoplasm in eukaryotes before translation is initiated.
The use of a bicistronic mRNA reporter in an in vitro translation extract allowed us to multiplex the assay, thus reducing our reagent cost and consumption of small molecule ligand collection. Herein, we have identified several novel protein synthesis inhibitors and characterized their activity profiles.
All cloned PCR products and oligonucleotide inserts were sequenced to ensure the absence of undesirable mutations. Sonenberg McGill University. In vitro Krebs translation extracts were prepared as follows. The cells were left to swell on ice for 20 min, transferred to a 40 ml type B Dounce homogenizer Bellco and lysed 20—40 strokes lysis was monitored by staining cells with Trypan blue and visual inspection by microscopy.
To determine the inhibitory effect of the compounds tested, data for each compound were internally normalized to the control mean values translations in the absence of compound on each plate and expressed as relative inhibition.
In vitro translation assays and secondary assay In vitro transcriptions were performed essentially as described previously For in vitro translations using [35S]methionine incorporation into endogenous protein, translations were set up as above except that the micrococcal nuclease treatment step was omitted. Radioactivity was determined by scintillation counting. Plates were then processed for luciferase measurements. The final KOAc concentration was adjusted to mM.
Centrifugation was for 3. The basis of the screen lies in the ability to quantitatively assess the products of the firefly FF and renilla Ren cistrons, following in vitro translation of the test transcript. A general inhibitor of translation initiation, elongation or termination would be expected to reduce both firefly and renilla luciferase production.
The strategy we have established for the multiplexed screen is outlined in Figure 1 C. At this point, compounds that inhibited in the secondary assay are considered candidate inhibitors. Assay validation To maximize the sensitivity of the in vitro translation system for detection of protein synthesis inhibitors using the bicistronic luciferase reporter several conditions first had to be met. For eukaryotic in vitro translation screens, we had the option of using several extracts, derived from wheatgerm, rabbit reticulocyte lysates, Krebs cells or HeLa cells.
Pelletier, unpublished data. In our small molecule screen we used extracts prepared from Krebs cells as these could be easily prepared in large quantities. The results indicate that for both firefly and renilla luciferase expression, this concentration range was within the linear range.
Since addition of m7GDP, but not GDP, to the reaction significantly altered the firefly to renilla expression ratio compare lane 3 with lanes 1 and 2 , these results indicate that translation of firefly luciferase in Krebs extracts is cap dependent. These parameters suggest the suitability of this assay for use in a miniaturized format.
To validate that this screen could identify translation inhibitors, we chose a set of known inhibitors representing examples of different activity spectra Supplementary Material, Fig.
The behavior of many of these compounds in Krebs extracts has not been previously documented. The mechanism of action of baccharinol has not been reported, but it is a trichothecin epoxide that likely inhibits elongation. The distribution of the firefly and renilla values obtained from the tested compound population indicated a good performance of this assay in the high throughput screen format.
Of these, five 0. The set of small molecule ligands still positive after the selection criteria denoted in Figure 1 C are listed in Table 1. Of the 36 compounds identified in our screen 0. These compounds provide an internal validation of the ability of our screen to identify inhibitors of protein synthesis.
Since the mechanisms of action of these compounds are well characterized, they were not further studied. DNA binding results in loss of negative supercoils producing a decrease in mobility when analyzed by agarose gel electrophoresis Fig. Since the initial high throughput translation assay was set up to identify compounds that could inhibit one of several steps, we undertook a series of experiments to deconvolute their mode of action. Malina and J. Given the large number of intercalators identified in this study, detailed characterization of their modes of action will be presented elsewhere A.
NSC maintained the same differential effect on firefly and renilla expression as observed in Krebs extracts Fig. To assess if any of these compounds exerted an effect on prokaryotic protein synthesis, they were tested in an E.
A compound that inhibits initiation of translation will prevent formation of 80S ribosome—mRNA complexes, whereas inhibitors of elongation or termination will not affect loading of ribosomes onto mRNA templates.
Ribosome-bound EF-2 is not susceptible to inactivation by the toxin. Puromycin is an analog of the terminal aminoacyl-adenosine part of aminoacyl- tRNA Figure Our results suggest that these compounds inhibit either elongation or termination of translation. Guenier and J.
For in vitro translations using [35S]methionine incorporation into endogenous protein, translations were set up as above except that the micrococcal nuclease treatment step was omitted.
Catalytic formation of a bond between the nascent polypeptide and puromycin is followed by the release of the peptidyl-puromycin from the ribosome, as no further elongation is possible. These results indicate a strong discrimination on the part of suramine, NSC and NSC for the eukaryotic translation apparatus. Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate. For example, eIF-4E is a protein that binds directly to the 7-methylguanosine cap Section
Thus, there is ample opportunity for the formation of complex secondary structures that must be removed to expose signals in the mature mRNA. Many Antibiotics Work by Inhibiting Protein Synthesis The differences between eukaryotic and prokaryotic ribosomes can be exploited for the development of antibiotics Table The toxic effects of ricin a protein present in the castor bean have been known for nearly a century. Included among these are a number of antibiotics produced by one strain of microorganism and lethal to other strains of the same or a different species.
Termination in eukaryotes is carried out by a single release factor, eRF1, compared with two in prokaryotes. In eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine. The tumor suppressor gene product pRB directly impacts on the translation process by affecting the levels of ribosomes for a review see 6. THchodermin is the only chemical compound so far identified as a specific inhibitor of the termination stage of polypeptide synthesis.
This may indicate the need to screen a larger, more diverse collection of compounds or alternatively may indicate that ligands binding to the CAG repeat were unable to provide a kinetic barrier to inhibit initiation. Our results suggest that these compounds inhibit either elongation or termination of translation. This antibiotic mimics aminoacyl-tRNA and binds to the free A site of ribosomes engaged in protein synthesis. Initiator tRNA. However, as in prokaryotes, a special tRNA participates in initiation.
This antibiotic mimics aminoacyl-tRNA and binds to the free A site of ribosomes engaged in protein synthesis. Plates were then processed for luciferase measurements. For example, eIF-4E is a protein that binds directly to the 7-methylguanosine cap Section The tumor suppressor gene product pRB directly impacts on the translation process by affecting the levels of ribosomes for a review see 6. The cumulative knowledge gained from studying inhibitors of translation demonstrates the power of applying a chemical biology approach to study this process. NSC inhibits initiation of protein synthesis, as it prevented the formation of 48S and 80S initiation complexes Fig.
Surprisingly, no report documents the systematic search for new inhibitors of eukaryotic protein synthesis, with the majority of compounds currently used having been identified several decades ago 1 , 2. The cumulative knowledge gained from studying inhibitors of translation demonstrates the power of applying a chemical biology approach to study this process. Eukaryotic ribosomes are larger.
However, as in prokaryotes, a special tRNA participates in initiation. Centrifugation was for 3. Many features of RNA make it an attractive target: it is central to many functions of the cell, contains complex secondary and tertiary structural folds and lacks a cellular repair mechanism. The majority of known protein synthesis inhibitors bind to ribosomes, with some of these making key contacts with the RNA component 8 — A compound that inhibits initiation of translation will prevent formation of 80S ribosome—mRNA complexes, whereas inhibitors of elongation or termination will not affect loading of ribosomes onto mRNA templates. Of these, five 0.
Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. For example, the antibiotic puromycin inhibits protein synthesis by causing nascent prokaryotic polypeptide chains to be released before their synthesis is completed. Pelletier, manuscript in preparation.
Tetracycline inhibits protein synthesis by blocking aminoacyl-tRNA binding to the small subunit. Search term Section The mechanism of action of baccharinol has not been reported, but it is a trichothecin epoxide that likely inhibits elongation. EF-2 exists in cells in two forms— ribosome-bound and free. Components of the translation apparatus are overexpressed or mutated in cancers 5.