Biomedical subjects
Publications and source records attributed to S L Bogdanova.
Cleavage of 16S ribosomal RNA (rRNA) from E. coli "hammerhead" type ribozymes as well as by RNAase iI in the presence of "hymeric" (2'-deoxy-F-thymidine containing) oligonucleotides has been studied. The conditions for the cleavage of a desired single internucleotide bond have been found for a large molecule with a very complicated secondary and three-dimensional structure.
Synthetic mRNA analogues were constructed with sequences related to the Cro-protein mRNA from lambda-phage and prepared by T7 transcription. Each mRNA contained several thiouridine (thio-U) residues. The regions upstream from the AUG initiator codon of the mRNA were the same in all the messages, whereas in the downstream part the thio-U residues were placed in selected positions. These positions covered the region from +4 to +16 (A in the initiator AUG codon being defined as +1). After binding to the ribosome in the presence of initiator tRNA the thio-U residues were activated by u.v. irradiation and the resulting sites of cross-linking to 16 S rRNA and ribosomal proteins were analysed. Cross-links to several ribosomal proteins were identified in different types of complex. Changes in the conformation of the small ribosomal subunit in different initiation and elongation complexes are discussed.
The role of the ribosomal protein S1 in translation of a mRNA containing an extended Shine-Dalgarno sequence has been investigated. Using the toe printing technique, the formation of a ternary initiation complex with both S1-depleted 30S subunits and subunits treated with anti-S1 antibodies and mini-mRNA containing an lambda-cro-mRNA translational initiation region with a very long (9 nucleotides) Shine-Dalgarno sequence, has been demonstrated. It is concluded that initiation of translation on mRNA with extended SD-sequence is S1-independent. By means of an E. coli cell-free system of translation (S-100 extract), the translation of mini-cro-mRNA by S1-depleted ribosomes has been shown. In contrast with mini-cro-mRNA, the 30S subunits without protein S1 are inactive both in the ternary initiation complex formation and in cell-free translation with MS2 or fr phage RNAs and RNA protein III of phage fd.
To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
The role of ribosomal protein S1 in the translation of mRNA containing an extended Shine-Dalgarno sequence was investigated. Using the toeprinting technique, formation of the ternary initiation complex between 30S subunits, both S1-depleted or treated with anti-S1 antibodies, and mini-mRNA containing the 9 nucleotide-long Shine-Dalgarno sequence was studied. It was concluded that the initiation of translation on mRNA with an extended Shine-Dalgarno sequence is S1-independent. It was demonstrated that S1-depleted ribosomes effectively translate the cro-mini-mRNA in a cell-free system. In contrast to cro-mini-mRNA, 30S subunits without protein S1 are inactive in ternary initiation complex formation with, and cell-free translation of, MS2 or fr phage RNAs and RNA protein III of phage fd.
A series of plasmids encoding native and modified sequences of the hepatitis B virus core antigen (HBcAg) was created. Analysis of the products generated by expression of the plasmid genomes in Escherichia coli showed that a polypeptide with primary structure identical to that deduced for native HBcAg forms particles in the bacterial cells which are indistinguishable from the native nucleocapsids in morphological and antigenic properties. Removal of the thirty-nine C-terminal amino acids which form a protamine-like domain caused insignificant impairment of the particle-forming process. Modification of the N-terminal region of the polypeptide showed that at least part of the structural determinant governing particle formation is localised between amino acid residues 3 and 11. When the plasmid genes were expressed in an E. coli cell-free transcription - translation system, polypeptides devoid of ten to twenty N-terminal amino acids were formed in addition to the full-length products. From the results obtained it is proposed that a protease digestion site situated within the region containing amino acid residues 10 - 20 plays a role in the formation of the HBe antigen.
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The synthesis of 5S rRNA and 4.5S RNA in E. coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied. The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B. When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain. Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells. At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors. The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation.
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Three cryptic plasmids have been isolated from cyanobacterium Synechocystis sp. 6803::pSS2 (1.4 Md), pSS3 (36 Md), pSS4 (60 Md). Plasmid DNA was isolated in Cs-Cl-EB density gradient and analyzed by gel electrophoresis and electron microscopy by gel electrophoresis and electron microscopy techniques. The restriction map is constructed for plasmid pSS2 having the cleavage sites for Sau3a, HincII, HindIII, MspI restriction endonucleases. The plasmid may be used to construct the recombinant vector DNAs capable of autonomous replication in cyanobacterium Synechocystis sp. 6803. cells.
A rapid method for mapping exposed cytosine residues in 5'-[32P]-labeled RNA molecules is suggested. The exposed cytosines (C's) are converted into uracyls (U's) by bisulphite treatment at pH 5.8 in the presence of Mg2+, followed by complete modification of the residual (non-exposed) C's by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary C leads to U conversion. The location of the exposed C's is determined by comparing the products of partial T1, T2, A and U2 ribonuclease digestions of the C leads to U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNATrp (yeast) which have been found in the anti-codon loop and at the 3'-end of the molecule. In tRNATrp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with what is known about exposed C's for other tRNAs.
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RRelationship between the level of induction of colicin E1 synthesis and the content of plasmid DNA in Escherichia coli K12S (E1) cells was studied. The number of Col E1 DNA copies per cell was found to decrease with growing generation time for different growth media and to increase continuously for the time of synchronous cell division cycle. No apparent relation of the level of spontaneous induction and of induction caused by N-methyl-N'-nitro-N-nitrosoguanidine to the number of Col E1 DNA copies per cell was observed. It is inferred that the content of plasmid DNA in the cells is not among the main factors determining the level of induction of colicin synthesis.
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