共查询到20条相似文献,搜索用时 31 毫秒
1.
Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides 总被引:2,自引:0,他引:2
New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA. 相似文献
2.
Mathias Sprinzl Karl-Heinz Scheit Hans Sternbach Friedrich von der Haar Friedrich Cramer 《Biochemical and biophysical research communications》1973,51(4):881-887
2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNAPhe by tRNA nucleotidyl transferase. tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNAPhe-C-C-3′dA proved to be chargeable. 相似文献
3.
Soria Baouz Anne Woisard Sylvie Sinapah Jean-Pierre Le Caer Manuela Argentini Konstantin Bulygin Gustave Aguié Codjo Hountondji 《Biochimie》2009,91(11-12):1420-1425
Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNAAspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNAAsp competed with tRNAAspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e. 相似文献
4.
Yuri V. Ivanov Eugen M. Saminsky 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,800(2):203-206
Puromycin inhibits the interaction of peptidyl-tRNA analogues AcPhe-tRNAox-redPhe, AcPhe-tRNAPhe and fMet-tRNAfMet with the donor (P-) site of Escherichia coli ribosomes. affects almost equally both the rate of the binding and the equilibrium of the system. This means that the effect is due to direct competition for the P-site, but not due to the indirect influence via the acceptor (A-) site. The inhibition was observed also in 30 S ribosomal subunits, therefore the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements show that the affinity of puromycin for its new ribosomal binding site is similar to its affinity for the acceptor site of the peptidyl transferase center. 相似文献
5.
An apparent conformational change in tRNA(Phe) that is associated with the peptidyl transferase reaction 总被引:3,自引:0,他引:3
Fluorescence techniques were used to detect changes in the conformation of tRNA(Phe) that may occur during the peptidyl transferase reaction in which the tRNA appears to move between binding sites on ribosomes. Such a conformational change may be a fundamental part of the translocation mechanism by which tRNA and mRNA are moved through ribosomes. E. coli tRNA(Phe) was specifically labeled on acp3U47 and s4U8 or at the D positions 16 and 20. The labeled tRNAs were bound to ribosomes as deacylated tRNA(Phe) or AcPhe-tRNA. Changes in fluorescence quantum yield and anisotropy were measured upon binding to the ribosomes and during the peptidyl transferase reaction. In one set of experiments non-radiative energy transfer was measured between a coumarin probe at position 16 or 20 and a fluorescein attached to acp3U47 on the same tRNA(Phe) molecule. The results indicate that the apparent distance between the probes increases during deacylation of AcPhe-tRNA as a result of peptide bond formation. All of the results are consistent with but in themselves do not conclusively establish that tRNA undergoes a conformational change as well as movement during the peptidyl transferase reaction. 相似文献
6.
7.
Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNAYPhe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNAPfPhe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNAYPhe or tRNAPfPhe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site. 相似文献
8.
D Richter 《Biochemical and biophysical research communications》1978,81(2):359-365
Crude stringent factor, prepared from a mutant strain with low levels of tRNA nucleotidyl transferase, synthesizes little or no (p)ppGpp in the presence of tRNAPhe-CpC; addition of yeast tRNA nucleotidyl transferase, however, fully restores (p)ppGpp formation, indicating that the complete CCA terminus of the tRNA molecule is a prerequisite in the (p)ppGpp synthesizing reaction. When the terminal purine is replaced by a pyrimidine base as in the case of tRNAPhe-CpCpC; or when the latter is extended by addition of AMP yielding tRNAPhe-CpCpCpA, both modified tRNAs are low in stimulating the (p)ppGpp synthesizing reaction. Hence activation of the stringent factor by tRNA requires (i) the terminal purine base and (ii) the precise fitting of the CCA terminus to the acceptor site of the ribosome. 相似文献
9.
Analysis of a drosophila tRNA gene cluster 总被引:23,自引:0,他引:23
10.
We isolated several mutants with nucleotide substitutions in alanine tRNA (tRNAAla) that resulted in glutamine tRNA (tRNAGli) acceptor identity in Escherichia coli. These substitutions were in three regions of tRNA structure not previously associated with tRNAGln acceptor identity. Only the phosphate-sugar backbone moieties of these nucleotides interact with the enzyme in the previously determined X-ray crystal structure of the complex between tRNAGln and glutaminyl-tRNA synthetase. We conclude that these sequence-dependent phosphate-sugar backbone interactions contribute to tRNAGln identity, and argue that the interactions help communicate enzyme recognition of the anticodon to the acceptor end of the tRNA and the catalytic center of the enzyme. 相似文献
11.
Valine specific transfer RNA (tRNAVal) was isolated from Bacillus stearothermophilus and Escherichia coli by chromatography on benzoylated DEAE–cellulose (BD–cellulose). Likewise isoleucine specific transfer RNA (tRNAIle) was isolated from B. stearothermophilus and from Mycoplasma sp. Kid. The thermal denaturation profiles (melting curves) of the two tRNAVal species in the presence of Mg+ + were nearly identical. However, the Tm for the Kid tRNAIle was about 10°C lower than that for the B. stearothermophilus tRNAIle. A nuclease and tRNA-free aminoacyl-tRNA synthetase (AA-tRNA synthetase) preparation from B. stearothermophilus was able to function efficiently at temperatures up to 80°C in the aminoacylation of all four tRNA species. Determination of the amino acid-acceptor activity of each tRNA species as a function of temperature of the aminoacylation reaction showed in each case a strong correlation between the loss of acceptor activity and the thermal denaturation profile of the tRNA. Evidence is presented that the loss in acceptor activity is most likely due to a change in structure of the tRNA as opposed to denaturation of the enzyme. These results further support the idea that correct secondary and/or tertiary structure must be maintained for tRNA to be active as a substrate for the AA-tRNA synthetase. 相似文献
12.
Kazuyuki Nagamatsu 《Journal of biomolecular structure & dynamics》2013,31(4):729-739
Abstract Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNAPhe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNAPhe was measured by a singlet-singlet energy transfer. Conformational changes of tRNAPhe with binding of tRNAGlu 2, which has the anticodon UUC complementary to the anticodon GAA of tRNAPhe, were investigated. The distance obtained at the ionic strength of 100 mM K+ and 10 mM Mg2+ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNAGlu 2 is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ55 in the TΨC loop of tRNAPhe, Stern-Volmer quenching experiments for the probe with or without added tRNAGlu 2were carried out. The results showed greater access of the probe to the quencher with added tRNAGlu 2. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNAGlu 2 and some structural collapse occurs at the corner of the L-shaped structure. 相似文献
13.
Yu. N. Vorobjev 《Molecular Biology》2005,39(5):777-784
Three-dimensional atomic models of complexes between yeast tRNAPhe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNAPhe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNAPhe destruction and formation of the final tRNAPhe-oligonucleotide complementary complex. The primary complex formation and the following tRNAPhe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element. 相似文献
14.
New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center. 相似文献
15.
Angela Wai Shan Fung Charles Chung Yun Leung Richard Peter Fahlman 《RNA (New York, N.Y.)》2014,20(8):1210-1222
Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNALeu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNALeu (CAG)—a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed. 相似文献
16.
17.
18.
19.
Zhi-Xuan Huang Jing Li Qing-Ping Xiong Hao Li En-Duo Wang Ru-Juan Liu 《Nucleic acids research》2021,49(22):13045
Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism. 相似文献
20.
Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNAiMet); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNASer, tRNATrp, and tRNALys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNAPhe, tRNA1Gly, tRNA2Gly); Class D contains tRNA which totally lacks ribothymidine (tRNAVal). Only those tRNAs in Class C are acceptable substrates for uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis. 相似文献