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P Szafrański

Publications and source records attributed to P Szafrański.

At least 19 recordsLinked to original sources
PubMed1992

On the evolution of the bacterial major sigma factors.

The existence of internal sequence homologies between the N-terminal halves of the gram-negative bacterial major sigma factors and their C-terminal halves, which correspond to minor factors, is reported. In the case of Escherichia-Salmonella sigma-70, an apparent homology was even found between the C-terminal helix-turn-helix DNA-binding motif and the corresponding region of the peptide N half, which, however, is not directly engaged in promoter recognition. It is proposed that major sigma factors may have originated by duplication and fusion of a DNA unit related to the ancestral gene for the whole sigma family. Coevolution of major sigma structures and complex promoters is suggested.

Amino Acid Sequence↗
PubMed1991

Role of metal ions in the assembly and decay of the transcription initiation complex on tRNA gene in yeast extracts.

Studies on formation of the transcription initiation complex on the tRNA(Tyr) gene in yeast extracts with the use of Sarkosyl-challenge assay proved that a single magnesium ion is indispensable for the complex assembly in the absence of nucleotides. Other cations do not substitute for Mg2+. The optimal KC1 concentration for transcription of the gene by the assembled initiation complex was found to be twice as high as that for the assembly process and close to that observed previously for the TFIIIC (or tau) factor dissociation from the promoter. The preformed complex remained stable for several hours at room temperature and its decay was not influenced by ionic strength. The data seem to support the notion that the TFIIIC factor is used only for assembly of the initiation complex and is not necessarily involved in the subsequent steps of transcription.

Genes, Fungal↗
PubMed1990

Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA.

Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per micrograms DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (ApT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-3' linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region.

Animals↗
PubMed1985

Substrate selection by RNA polymerase from E. coli. The role of ribose and 5'-triphosphate fragments, and nucleotides interaction.

Steady-state kinetic studies of the rifampicin-effected abortive initiation of transcription by E. coli RNA polymerase (EC 2.7.7.6) on the A1 T7 phage promoter were carried out with the use of ATP, UTP and a number of their appropriately modified analogues. The kinetic parameters KiA, KmB, Ki and KsB characterizing the affinity of the substrates and inhibitors of the reaction to the initiation and elongation sites of the enzyme:promoter and the enzyme:promoter:nucleoside triphosphate complexes were determined therefrom. Their comparative analysis indicated that 1) the triphosphate chain of the initiating purine nucleoside triphosphate interacts with some protein acceptor groups through the alpha- and beta-phosphate residues; the phosphates are engaged in binding of nucleoside triphosphates at the elongation site in the absence of the primer nucleotide; 2) the ribose 2'-OH of the elongating nucleotide, but neither of the ribose hydroxyl groups of the initiating nucleotide, participate in substrate recognition by protein receptors; 3) either substrate, ATP or UTP, bound to the initiation complex increases by about the same factor (greater than or equal to 10) the affinity of the other to its binding site; 4) the 3'-OH of the primer nucleotide and the gamma-phosphate of the elongating nucleotide are involved in the synergistic interaction of the substrates; alpha- and beta-phosphates of the elongating nucleotide, bound to some protein receptors, also contribute to this process. It is postulated that the interaction of substrates is mediated through an Mg2+ ion, known to be required for binding of the substrates in the elongation site, and a minimal molecular model of a PuoTP:Mg (II): nucleoside triphosphate chelate complex in the catalytic centre of the transcription initiation open complex is proposed.

Adenosine Monophosphate↗
PubMed1982

Effect of secondary structure of messenger ribonucleic acid on the formation of initiation complexes with prokaryotic and eukaryotic ribosomes.

The effect of modification of the secondary structure of phage f2 RNA and brome mosaic virus (BMV) RNA 3 on the formation of initiation complexes in Escherichia coli and wheat germ protein-synthesizing systems was studied. Modification of the RNAs was achieved by using O-methylhydroxylamine, which specifically reacts with cytosines; this leaves the initiation codons unchanged and, under denaturing conditions, leads to irreversible unfolding of the RNA. E. coli ribosomes interact with newly exposed AUG/GUG codons in the modified templates forming polysomes, whereas they form monosomes with native f2 RNA or BMV RNA 3. With wheat germ ribosomes, disomes are formed in the presence of BMV RNA 3, either native or modified. With f2 RNA, eukaryotic ribosomes form monosomes, independent of the secondary structure of the template. The results indicate that, in contrast to prokaryotic ribosomes, binding of eukaryotic ribosomes to f2 RNA or BMV RNA 3 is not affected by modification of the secondary structure of these messengers.

Cells↗
PubMed1977

The effect of modification of cytosines in Escherichia coli 16-S rRNA on reconstitution and function of 30-S ribosomes.

O-Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16-S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16-S rRNA chain. Preparations of methoxyaminated 16-S rRNA, containing 2--130 modified cytosines/chain, still retained the ability to bind 30-S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16-S rRNA. The particles obtained sedimented slower than native 30-S subunits, lost their ability to associate with 50-S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16-S rRNA did not affect binding of poly(U) by reconstituted particles.

Cytosine↗
PubMed1976

Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis.

1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.

Cell-Free System↗
PubMed1975

Recognition of initiation codons in modified f2 RNA by Escherichia coli ribosomes.

f2 phage RNA treated with O-methylhydroxylamine under denaturing conditions loses its ordered structure with consequent exposure of the normally hidden initiation codons. In the presence of Escherichia coli ribosomes and crude initiation factors modified f2 RNA binds about 50 times more f-[3H]Met-tRNA than native f2 RNA. The interaction of native f2[14C]RNA with ribosomes requires initiation factors. The binding of O-methylhydroxylamine-modified f2 [14C]RNA to E. coli 70-S or 20-S ribosomes does not depend on the presence of initiation factors. A significant number of ribosomes deficient in initiation factors interact with a molecule of modified f2 [14C]RNA. Treatment of the resultant polysomal complex with pancreatic RNase yields ribosomes with f2 RNA fragments protected against RNase. Almost all AUG/GUG codons in the f2 RNA are located on the RNase-insensitive ribosome-bound fragments, constituting only 25% of the entire molecule. Addition of crude initiation factors to such ribosomes with fragments of modified f2 RNA promotes binding of f-[3H]Met-tRNA. The resultant complex is fully reactive with puromycin. No binding of Ac-Phe-tRNA takes place under similar conditions.

Codon↗
PubMed1975

Preparation and characteristics of the structure of methoxyamine-modified f2 RNA.

Treatment of phage f2 RNA with [14-C]methoxyamine under non-denaturing conditions resulted in modification of exposed cytosines only. On methoxyamine treatment in the presence of 6 M-guanidine, all cytosines were modified. Under the conditions applied, no modification of adenine base in RNA chain occurred. The structure of modified f2 RNA preparations was studied by melting and sedimentation analysis. The ratio between the modification products (N-4-methoxycytosine and N-4-methoxy-6-methoxyamino-5,6-dihydrocytosine) was determined in RNA preparations modified under non-denaturing and denaturing conditions.

Binding Sites↗