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D M Graĭfer

Publications and source records attributed to D M Graĭfer.

At least 19 recordsLinked to original sources
PubMed1991

[Segments of 18S rRNA, located in the area of the mRNA-binding center of ribosomes from human placenta].

The affinity labelling of human placenta 80S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoramide of hexauridylate has been studied. This mRNA analogue has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of the cognate tRNA(Phe). Incubation of the mRNA analogue in the complex with ribosomes and Phe-tRNAPhe) leads to its covalent attachment exclusively to the small subunit (mainly to 18S rRNA). The reaction site has been shown by hybridisation experiments to be located within positions 975-1055 of 18S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.

Affinity Labels↗
PubMed1987

[Preparation of Escherichia coli 70S ribosomes labeled with 35S].

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.

Escherichia coli↗
PubMed1987

[Interaction of s4U8 region of tRNA Phe with tRNA-(adenine-1-)-methyltransferase from Thermus thermophilus].

Photoaffinity labelling of tRNA (adenine-1-)-methyltransferase with an E. coli tRNA(Phe) derivative bearing 4-azidophenylmercuro group attached to s4U residue as well as direct photocross-linking of the native tRNA(Phe) with the enzyme via s4U residue has been studied. Both techniques labelling gave similar results, leading to covalent attachment of tRNA(Phe) to the enzyme within a specific complex. The data obtained indicate unambigously that s4U residue contacts with tRNA (adenine-1-)-methyltransferase within the corresponding specific complex.

Cross-Linking Reagents↗
PubMed1987

[The use of cross-linking platinum reagents in the study of tRNA and mRNA interaction with ribosomes].

The use of some bifunctional Pt(II)-containing cross-linking reagents for investigation of structural organization of ribosomal tRNA- and mRNA-binding centres is demonstrated for various types of [70S ribosome.mRNA-tRNA] complexes. It is shown that treatment of the complexes [70S ribosome.Ac[14C]Phe-tRNA(Phe).poly(U)], [70S ribosome.3'-32pCp-tRNA(Phe).poly(U)] and [70S ribosome.f[35S]Met-tRNA(fMet).AUGU6] with Pt(II)-derivatives results in covalent attachment of tRNA to ribosome. AcPhe-tRNA(Phe) and 3'-pCp-tRNA(Phe) bound at the P site were found to be cross-linked preferentially to 30S subunit. fMet-tRNA(fMet) within the 70S initiation complex is cross-linked to both ribosome subunits approximately in the same extent, which exceeds two-fold the level of the tRNA(Phe) cross-linking. All used tRNA species were cross-linked in the comparable degree both to rRNA and proteins of both subunits in all types of the complexes studied. 32pAUGU6 cross-links exclusively to 30S subunit (to 16S RNA only) within [70S ribosome.32pAUGU6.fMet-tRNA(fMet)] complex. In the absence of fMet-tRNAfMet the level of the cross-linking is 4-fold lower.

Chemical Phenomena↗
PubMed1986

[Alkylation of tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of d(ATTTTCA)].

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of oligonucleotide d(ATTTTCA) complementary to the sequence UGAAms2i6AA psi in the anticodon loop of tRNAPhe was studied. Three guanine residues--G28/29, G24 and G10 were found to be alkylated. Two binding sites for the reagent in the tRNA were assumed to be present. The efficiency of the alkylation of tRNA from these sites as well as an average association constant (Ka 3,8 X 10(3)M-1) for the reagent interaction with tRNA were evaluated.

Alkylation↗
PubMedDate not supplied

[Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit].

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

Hepacivirus↗
PubMedDate not supplied

[Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives].

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable group at different positions. A comparison of data on template position on the human and Escherichia coli ribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A, P, and E sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem-loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.

Codon↗
PubMedDate not supplied

[Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes].

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.

Azides↗
PubMedDate not supplied

[The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU].

Three mRNA analogs--derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position--were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA(Phe) was used, while tRNA(Val) was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions -3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with soft UV light (lambda > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions -1 to +3 binds to G1207, while that in positions -2 or -3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the -3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the -3 position, this crosslinking was not observed in the absence of tRNA.

Codon↗
PubMedDate not supplied

[Highly conserved region 1816-1831 of the 18S ribosomal RNA is close to the first nucleotide of the A-site codon in elongation and termination of translation].

Two mRNA analogs, pUUCUAAA (with stop codon UAA) and pUUCUCAA (with Ser codon UCA) containing a perfluoroarylazido group at U4, were used to study the position relative to the 18S rRNA for the first nucleotide of the codon located in the A site of the human 80S ribosome. To place UAA or UCA in the A site, UCC-recognizing tRNAPhe was bound in the P site. With each analog, crosslinking was detected for highly conserved fragment 1816-1831, which contains invariant dinucleotide A1823/A1824 and is in helix 44 at the 3' end of the 18S rRNA. Since 18S rRNA modification did not depend on whether the U4 photoreactive group was in the sense or stop codon, it was assumed that polypeptide chain release factor 1 directly recognizes the trinucleotide of a stop codon located in the A site.

Base Sequence↗
PubMedDate not supplied

[Affinity modification of 80S ribosomes from human placenta by derivatives of tri- and hexauridylates as mRNA analogs].

Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit. The sites of the reagents attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 derivative and within 976-1164 for (pU)3 one. These sites are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

Alkylating Agents↗
PubMedDate not supplied

[Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with alkylating groups at the 3'-end].

2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene derivatives of oligoribonucleotides (Up)5U[32P]pC and (Up)11U[32P]pC were used for affinity labeling of 80S ribosomes from human placenta in the presence of cognate Phe-tRNA(Phe). The derivatives retained the ability of tRNA(Phe)-dependent binding with 80S ribosomes and within the corresponding complexes were cross-linked to 40S subunits (preferentially to 18S rRNA). Sites of the mRNA analogs attachment were identified by blot-hybridization of the modified rRNA with restriction fragments of corresponding rDNA. They are located within positions 1610-1747 and 1748-1865 for both analogs. These fragments are situated at the 3'-end region of 18S rRNA in a highly conserved part of eukaryotic small subunit rRNA secondary structure.

Affinity Labels↗
PubMedDate not supplied

[Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives].

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, UCC-recognizing tRNA(Phe) was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 or nucleotide A1825 in helix 44 close to the 3' end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem-loop segment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in various organisms.

Base Sequence↗
PubMedDate not supplied

[Approach to identifying the functionally important segments of RNA, based on complementation-addressed modification].

An approach based on complementation-addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5'-P and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect, whereas alteration of hairpin IIIe substantially reduced the binding. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play a critical role in the binding of HCV IRES with the 40S subunit.

Base Sequence↗