TRNA analog

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

In another case, two ribosomal proteins at the peptidyl transferase site were derivatized with photolabile aminoacyl-tRNA analogs (Hsiung et al., 1974 Hsiung and Cantor, 1974). 

Puromycin is a specific metabolic inhibitor of protein synthesis and acts as an aminoacyl-tRNA analog and peptidyl acceptor. The latter causes premature chain... 

The protein synthesis inhibitors tetracycline, chloramphenicol, and streptomycin all block bacterial protein synthesis. Several eukaryotic translational inhibitors have also been found and they include diphtheria toxin, ricin, and cycloheximide. Puromycin causes premature chain termination in both prokaryotes and eukaryotes by functioning as an aminoacyl tRNA analog. 

Some Yields of Covalent Reacitons between 70 S Ribosomes and tRNA Analogs ... 

Binding of Phe-tRNA derivatives was usually performed in media buffered at pH 7.2 to 7.5 and containing, Mg at 10-30 mM, K or NH/ at 0.03-0.15 M, and poly(U). With initiator tRNA analogs, the mRNAs employed were R17-RNA, f2-RNA, or T4-mRNA, Mg- concentration was reduced to about 5 mikf, and the mixtures were also supplemented with initiation factors and GTP. Concentration ratios of affinity labeling probes to ribosomes (70 S) varied from about 1.0 to large... 

Another characterization of the binding of peptidyl-tRNA analogs to the ribosome is based on the response to antibiotic inhibitors of peptidyl-transferase activity. Although Oen et al. observed no inhibition of reversible binding of iV-bromoacetyl Phe-tRNA by chloramphenicol, lin-comycin, or streptogramin A, the same antibiotics as well as erythromycin significantly inhibited the binding of p-azido-A-tBoc-Phe-Phe-tRNA. It should be noted, however, that in both cases the antibiotics markedly reduced the extent of the covalent reaction of the aflSnity probes with the ribosome. 

Photoreaction with A-(ethyl-2-diazomalonyl)Phe-tRNA does not inactivate peptidyltransferase. This observation agrees with results of affinity labeling experiments with other reactive peptidyl-tRNA analog these also suggest that the fixed A-acyl-aminoacyl-tRNA is able to participate in at least one peptidyl transfer. Incubation of the modified complex with Phe-tRNA yields an irreversible fixation of one to two additional phenylalanyl-groups to 23 S RNA. 

These peptidyl-tRNA analogs bind reversibly, in the presence of poly (U), to 70 S ribosomes and attach covalently upon irradiation to a specific site(s) on the 23 S RNA of the 50 S subunit. 

The series of compounds described here is suitable for screening the ribosomal components involved in peptidyltransferase activity. All compounds described are peptidyl-tRNA analogs, and the shortest member... 

Each tRNA has a different sequence at the anticodon loop that is complementary to the codon sequence in the RNA. The recognition structure that is formed is analogous to double-stranded, antiparallel DNA. If the codon (in the RNA) is GCA (written 5 to 3 ), the anticodon loop in the tRNA would have the sequence UGC (again written to 5 to 3 )-... 

The synthesis in many extant organisms of these two amide residues from their respective precursors glutamate and aspartate esterified to tRNA (the indirect aminoacylation pathways described in Sections 5.14.3 and 5.14.4) and that of other amino acid residues, such as selenocysteine (which is also synthesized from a precursor esterified on a tRNA °) support the model of prebiotic metabolism taking place at the surface of solid particles, " analogous to ancestral RNAs. 

The improvements in suppression efficiency that the Dougherty and Schultz groups were able to realize through use of tRNAs derived from different natural tRNA species confirm the importance of the tRNA sequence and structure in the suppression mutagenesis system. The results must be viewed with caution, however, as the efficiency of suppression is subject to other variables and is not fully understood. In different proteins, and at different sites within a protein, the results can be dramatically different. Nevertheless, it appears that use of these improved tRNAs will widen the range of analogs that can be introduced and increase protein yields. 

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

Fig. 11. Comparison of the peptidyl transfer reaction in the ribosome and in the selected peptidyltransferase ribozyme. The ribosome contains a binding site for the peptidyl-tRNA (P-site) and for the aminoacyl-tRNA (A-site). In the selected ribozyme the binding site for the AMP-Met-Bio substrate would be analogous to the P-site. The attacking a-amino group which is bound in the A-site in the ribosome is covalently attached to the 5 -end in the ribozyme. Catalytically active RNAs preferentially attach the biotin tag onto themselves and can thus be separated from the inactive ones...

Remarkably, incorporation of fluorinated amino acids into proteins can also be accomplished in vivo. This supposes that the fluorinated amino acid analogs are recognized by the appropriate amino acyl-tRNA synthetase enzyme with efficiency similar to that of the natural amino acid. The proliferase response elicited by a fluorinated analog (a trifluoroisoleucine derivative) of murine interleukin-2 produced in an appropriate Escherichia coli strain was nearly as high as that of the authentic cytokine, indicating folding into an authentic, native structure [84],... 

Emetine and dehydroemetine are natural alkaloid obtained from Cephaelis ipecacuanha and synthetic analog respectively. They are effective against tissue trophozoites of . histolytica. It has no effect on cysts but effective in amoebic liver abscess also. It acts by inhibiting protein synthesis by arresting intraribosome translocation of tRNA-amino acid complex. Dehydroemetine is less toxic than emetine and very effective drug for tissue amoebiasis. It is more rapidly eliminated from the body than emetine. 

The elongation cycle in eukaryotes is quite similar to that in prokaryotes. Three eukaryotic elongation factors (eEFla, eEFljSy, and eEF2) have functions analogous to those of the bacterial elongation factors (EF-Tu, EF-Ts, and EF-G, respectively). Eukaryotic ribosomes do not have an E site uncharged tRNAs are expelled directly from the P site. 

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