Biomedical subjects

A Kopylov

Publications and source records attributed to A Kopylov.

3 recordsLinked to original sources

PubMed2002

A comparative thermodynamic study for both natural and artificial RNA/DNA-protein binary complexes.

In recent years, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique has been developed into a fast growing field. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value both for the therapy and biosensors. A comparative study of thermodynamic for both natural and artificial RNA/DNA-protein complexes would establish bases for a specificity of complex formation. In particular, we have shown that aptamers could be used for a direct measuring of thrombin enzymatic activity in a solution.

Amino Acid Sequence↗

PubMed1992

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed. Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532. Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case. The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA. In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced. A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6. Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome.

Base Sequence↗

PubMed1991

The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.

Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex. The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.

Base Sequence↗