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2 edition of Chemical Modifications of the 50S ribosomal petidyl transferase activity found in the catalog.

Chemical Modifications of the 50S ribosomal petidyl transferase activity

Kwong Kee Wan

Chemical Modifications of the 50S ribosomal petidyl transferase activity

by Kwong Kee Wan

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  • 19 Currently reading

Published .
Written in English


The Physical Object
Pagination1 v., 149 leaves
Number of Pages149
ID Numbers
Open LibraryOL21265342M

Ribosomal RNA (rRNA) from all organisms contains modifications, and there is a correlation between the overall complexity of an organism and the number of modified nucleosides in its rRNA. The rRNA of the most primitive bacteria, such as some Mycoplasma species, may possess only 14 modified nucleosides (de Crécy-Lagard et al., ). The main finding is that ribosomes carrying a 2′-deoxyribose at A showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A had only little impact on peptide bond .

  These modifications could improve binding and result in the inhibition of the peptidyl transferase activity. One of the few macrolides able to inhibit peptidyl transferase is tylosin.   It has been known for 35 years that the peptidyl transferase activity responsible for the peptide bond formation that occurs during messenger RNA (mRNA)–directed protein synthesis is intrinsic to the large ribosomal subunit (), and it has been understood for even longer that the ribosome contains proteins as well as bacteria, for example, the large ribosomal subunit contains ∼

localize the 50S ribosomal subunit components directly in-volved in peptidyl transferase activity, by the method of affinity labeling. Theantibiotic chloramphenicol wasselected forthis purposein viewofits specific interaction withthe50S ribosomal subunit at, or in the vicinity of, the active site of peptidyl transferase (1, 2). An analog of. Miskin R, Zamir A, Elson D. Inactivation and reactivation of ribosomal subunits: the peptidyl transferase activity of the 50 s subunit of Escherihia coli. J Mol Biol. Dec 14; 54 (2)– Muth GW, Ortoleva-Donnelly L, Strobel SA. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center. Science.


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Chemical Modifications of the 50S ribosomal petidyl transferase activity by Kwong Kee Wan Download PDF EPUB FB2

Footprinting experiments. Ribosome isolation, chemical modification with dimethyl sulfate (DMS) and 1-cyclohexyl(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT), and primer extension procedures were carried out essentially as described previously ().Kethoxal modifications were performed by incubating 5 pmol of the antibioticS complexes for 30 min at 37°C in 50 μl Cited by: Abstract.

Extraction with 2 M lithium chloride removes a group of proteins (LiC1 SP) from 50S ribosomal subunits. Both the LiC1 SP and the resulting cores, which contain the remaining proteins as well as 5S and 23S RNA, lack peptidyl transferase activity, as measured by the "fragment reaction".

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both Cited by: Miskin R, Zamir A, Elson D.

Inactivation and reactivation of ribosomal subunits: the peptidyl transferase activity of the 50 s subunit of Escherihia coli. J Mol Biol. Dec 14; 54 (2)– Muth GW, Ortoleva-Donnelly L, Strobel SA. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.

by: Notably, the IC 50 values for molecules 2 and 8 were and μM, respectively. These values are superior to the IC 50 for chloramphenicol ( μM), a broad-spectrum antibiotic that binds the PTC, thereby inhibiting ribosome activity (Fig. 3B and Table S4†).

Peptidyl transferase activities of gapped-cp-reconstituted subunits containing 2′-deoxyribose modifications at A using CC-puromycin as acceptor substrate. (A) The reaction between N -acetyl-Phe-tRNA and [ 32 P]CC-puromycin was carried out for min, a time point that corresponded to the endpoint of the reaction catalyzed by.

The rate of reaction varied within a factor of 50– depending on the length of the peptidyl moiety of the P site tRNA, the C-terminal amino acid of the peptide, or the identity of the tRNA in the P site: the reaction rate was close to 1 s −1 with fMet-tRNA fMet in the P site, 10–20 s −1 with di- and tripeptidyl-tRNAs fMetPhe-tRNA Phe.

The recent publication of the high-resolution crystal structure of the Haloarcula marismortui 50S ribosomal subunit has placed A at the center of attention, as it was shown to be the closest nucleotide to a peptidyl transferase inhibitor (C-C-dA-phosphoramide-puromycin) designed to mimic the tetrahedral intermediate formed during transpeptidation (15–17).

Peptidyl transferase activity was measured essentially as by Cerna et al. 5 pmol of ribosomes were incubated with 5 pmol of N-acetyl-[ 14 C]Phe-tRNA Phe (provided by Professor S. Kirillov), 5 μg of poly(U) (Sigma), and the antibiotic to be investigated in μl of 50 mM Tris.

The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the peptidyl transferase center residing in the large ribosomal subunit. The linker region is essential for NTP-dependent inhibition of ribosomal peptidyl transferase activity by the NTP hydrolysis-incompetent VgaA LC EQ 2 mutant.

The NTPase-incompetent VgaA LC EQ 2 mutant does not rescue the puromycin reactivity of S. aureus 70S IC(MF) inhibited by 1 μM lincomycin (dark blue filled circles), and compromises. In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytically active 50S ribosomal subunits with an efficiency only 3−4-fold lower than that of natural 23S rRNA.

Thus, post-transcriptional modifications in 23S rRNA are not essential for the assembly or function of the 50S subunit of the ribosome.

This reconstitution sytem has been used to characterize. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these.

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification. Journal of Molecular Biology(1), DOI: /(79) Zvi Vogel, Miriam Altstein. The effect of puromycin on the biological activity of Leu-enkephalin. Peptidyl transferase activity was measured essentially as by 3; 5 pmol of ribosomes were incubated with 5 pmol of N‐acetyl‐[14 C]Phe‐tRNA Phe (provided by Professor S.

Kirillov), 5 µg of polyU (Sigma) and the antibiotic to be investigated in µl of 50 mM Tris‐HCl (pH ), mM KCl and 10 mM MgCl 2 for 30 min at 0°C. The drug. tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis.

Two regions resemble the peptidyl-transferase region of 23s ribosomal RNA in sequence and structural context; these. Dohme F, Fahnestock SR. Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification. J Mol Biol.

Mar 25; (1)– Dohme F, Nierhaus KH. Role of 5S RNA in assembly and function of the 50S subunit from Escherichia coli. Proc Natl Acad Sci U S A. Jul; 73 (7)–   Type B streptogramins act on the 50S ribosomal subunit in a similar fashion as the macrolides and compete for the same binding site.

The S B do not affect the peptidyl transferase reaction, but inhibit elongation after a few cycles of peptide bond formation; by analogy with the macrolides, S B are presumed to bind within the tunnel and block the path of the nascent polypeptide.

RNAs from a randomized pool were selected by affinity elution for binding to the molecule CCdApPuro, a high-affinity ligand of ribosomal peptidyl transferase designed as a transition-state analogue of peptide formation. The selected RNAs show affinity for CCdApPuro comparable to that of the peptidyl transferase center itself (Kd ≈ 10 nM).

Ferdinand Dohme, Stephen R. Fahnestock, Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification, Journal of Molecular Biology, /(79),1, (), ().

Peptidyl transferase is a ribozyme that facilitates formation of peptide bonds during translation. As an aminoacyltransferase, In this case, an amino acid chain is the functional group transferred by a peptidyl transfer involves the Transferase deficiencies are at the root of many common illnesses.

The most common result of a transferase deficiency is a.Peptidyl Transferase Protein tion for 30 min at 15, rpm (SS rotor, Sorvall RC-2B centrifuge). Pellets wereresuspendedinactivation buffer at a concentration of about 50 A units/ml.

Peptidyl Transferase Assay. The peptidyl transferase activity of particles was assayed by the method of Monro (10), after the particles were heated for.The 23S rRNA is a nt long (in E. coli) component of the large subunit of the bacterial/archean ribosomal peptidyl transferase activity resides in domain V of this rRNA, and this domain is the most common binding site for antibiotics that inhibit translation.

A well-known member of this antibiotic class, chloramphenicol, acts by inhibiting peptide bond formation, with recent.