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PubMed2006

Regiospecificity of the peptidyl tRNA ester within the ribosomal P site.

The ribosomal peptidyl transferase center is expected to be regiospecific with regard to its tRNA substrates, yet the ester linkages between the tRNA and the amino acid or peptide are susceptible to isomerization between the O2' and O3' hydroxyls of the terminal A76 ribose sugar. To establish which isomer of the P site tRNA ester is utilized by the ribosome, we prepared two nonisomerizable transition state inhibitors with either an A76 O2' or O3' linkage. Strong preferential binding to the O3' regioisomer indicates that the peptidyl transferase proceeds through a transition state with an O3'-linked peptide in the P-site.

Binding Sites↗
PubMed2005

An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.

The large ribosomal subunit catalyses the reaction between the alpha-amino group of the aminoacyl-tRNA bound to the A site and the ester carbon of the peptidyl-tRNA bound to the P site, while preventing the nucleophilic attack of water on the ester, which would lead to unprogrammed deacylation of the peptidyl-tRNA. Here we describe three new structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with peptidyl transferase substrate analogues that reveal an induced-fit mechanism in which substrates and active-site residues reposition to allow the peptidyl transferase reaction. Proper binding of an aminoacyl-tRNA analogue to the A site induces specific movements of 23S rRNA nucleotides 2618-2620 (Escherichia coli numbering 2583-2585) and 2541(2506), thereby reorienting the ester group of the peptidyl-tRNA and making it accessible for attack. In the absence of the appropriate A-site substrate, the peptidyl transferase centre positions the ester link of the peptidyl-tRNA in a conformation that precludes the catalysed nucleophilic attack by water. Protein release factors may also function, in part, by inducing an active-site rearrangement similar to that produced by the A-site aminoacyl-tRNA, allowing the carbonyl group and water to be positioned for hydrolysis.

Acylation↗
PubMed2006

The hybrid state of tRNA binding is an authentic translation elongation intermediate.

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.

Escherichia coli↗
PubMed1975

Cell-free synthesis of human interferon.

With mRNA prepared from induced human fibroblasts biologically active human interferon was synthesized de novo in a cell-free extract from mouse cells. The identity of the antiviral activity as human interferon was demonstrated by its species and antigenic specificity.

Animals↗
PubMed1991

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.

Base Sequence↗
PubMed1998

Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains.

It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23S ribosomal RNA, whereas domain VI could enhance the activity in trans. These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.

Catalysis↗
PubMed1985

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.

Acetates↗
PubMed1979

Studies on termination of protein synthesis in wheat germ.

Oligophenylalanines are soluble in m-cresol, but oligophenylalanyl-tRNAs are not. This differential solubility can be used to assay oligophenylalanines released from tRNA during their synthesis by wheat germ extracts. When poly U is the message, virtually no free product appears. When poly A U (A < U) is used, a considerable amount of oligophenylalanines are released. The fraction of product released is approximately constant with time, implying that a steady-state is not achieved between initiation and release. The dependence of release on various reaction variables and the effects of several inhibitors on release indicate that the reaction is probably catalyzed by peptidyl transferase, in accord with the mechanisms described for mammals and prokaryotes.

Cell-Free System↗
PubMed1977

[Acquired colour-vision-deficiencies caused by side-effects of pharmacotherapy (author's transl)].

Acquired colour-vision deficiencies are an early indicator for drug-induced retinopathy as well as drug-induced retrobulbar neuritis. Koellner's rule, which says, that damage of the retina induces a tritan-defect, and damage of the optic nerve induce a red-green-defect is also valid for defects secondary to drug-toxicity. Pseudoisochromatic plates, anomaloscope and other tests (Panel D-15-test) have to be selected correspondingly to use them as screening-methods.

Antipsychotic Agents↗
PubMed1988

Messenger RNA loses the ability to direct in vitro peptide synthesis following incubation with cisplatin.

Inhibition of in vitro translation activity by the chemotherapeutic agent cisplatin has been studied. Peptide synthesis was measured in translation assays prepared from guinea pig and rabbit reticulocyte lysates. There is a concentration- and time-dependent inactivation of translation by cisplatin. Inhibition is nearly maximal after a 30-min exposure. Translation is inhibited by cisplatin at concentrations that have been found after the administration of therapeutic doses. Evidence suggests that the suppression of peptide synthesis is due to an interaction between cisplatin and mRNA. An apparent qualitative difference in the peptide products translated from mRNA exposed to cisplatin is demonstrated. Large peptide products are inhibited to a greater extent than products with faster electrophoretic mobility. Decreased translation activity cannot be explained by digestion of the message by either cisplatin itself or by some contaminant of the drug solution.

Animals↗
PubMed1982

The elongation factor 1 from wheat germ: structural and functional properties.

Three forms of elongation factor 1 (EF1A, EF1B and EF1C) were isolated from wheat germ; the preparation obtained by successive chromatography on DEAE-Sephadex A-50, hydroxylapatite and Sephadex G-200 showed about 90% purity. The molecular masses of these forms were about: 61 000, 48 000 and 12 500, respectively. EF1A was the only form which formed the ternary complex GTP-EF1-AA-tRNA and was the most active in binding of Phe-tRNA to the poly-U programmed ribosomes. Based on the data of amino acid analysis, N-terminal amino acid determination and previously described proteolysis (Pulikowska et al., Biochem. Biophys. Res. Commun., 1979, 91, 1011-1017), it is assumed that EF1C is a basic structural subunit of elongation factor EF1 from wheat germ, but only EF1A shows the conformation-dependent full biological activity.

Amino Acids↗