Affinity Labeling of RNA Polymerase II in the Transcriptionally Active Complex by a Phosphorylating Analog of the Initiation Substrate

RNA polymerase II is composed of 12 subunits Substrate derivatives with various chemical groups were used to study the active site of RNA polymerase II. Reagents with arylazide groups were activated by irradi ation at the appropriate wavelength. An additional reagent was required for detection of the products of pro tein interaction with other reagents. For example, when carbonyl containing reagents were used for irreversible attachment of an affinity reagent to the protein, the gen erated Schiff bases were reduced with NaBH 4 . These studies were performed with highly purified enzyme preparations , poly[d(A T)] ACCELERATED PUBLICATION 0006 2979/00/6510 1129$25.00 ©2000 MAIK "Nauka / Interperiodica" * To whom correspondence should be addressed. Vol. 65, No. 10, 2000, pp. 1129 1134. Translated from Biokhimiya, Vol. 65, No. 10, 2000, pp. 1334 1340. Original Russian Text Copyright © 2000 Abstract-Affinity modification of RNA polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5′ terminal phosphate group with a 4 N,N dimethylaminopyridine residue (DMAP pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA polymerase II from modification. The third subunit may be involved in the formation of the substrate binding site of the enzyme

Prediction and verification of the influence of the rs367781716 SN P on the interaction of ТАТА -binding protein with the promoter of the human АВСА9 gene

The high-throughput sequencing project “1 000 Genomes” made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. Analysis of SNP markers (significantly frequent differences of individual genomes of patients from the reference human genome) allows physicians to optimize treatment. On the other hand, tens of millions of unannotated SNPs correspond to a gigantic number of false positive (false negative) candidate SNP markers that are selected by computer methods for comparison of their frequency in patients with that in healthy people. This approach contributes to undervaluation of clinically relevant SNPs and to unnecessary computational expenses (on verification of neutral SNPs). Preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, we found, using the SNP_TATA_Comparator web service, the unannotated SNP rs367781716: the substitution of ancestral T (health) with minor C at position –37 before the transcription initiation site of the АВСА9 gene. This SNP significantly reduces affinity of TATAbinding protein (TBP) for this gene’s promoter and corresponds to a deficiency (low protein level) of the АВСА9 gene product (the transporter ATP-binding cassette A9) in patients with the –37C allele. For preclinical empirical verification of rs367781716, we used an electrophoretic mobility shift assay (EMSA) to measure the rates of formation (ka) and decay (kd) of the complexes of TBP with an oligonucleotide matching either allele –37C or –37T of the АВСА9 gene. We found that the rate of formation (ka) of the TBP/TATA complex for the minor allele is 2.4-fold lower than that for the ancestral allele. We calculated the empirical value of the change in the equilibrium constant of dissociation (KD = kd /ka), which characterizes binding affinity of TBP for a promoter containing the ТАТА box. This empirical value matched the value predicted by SNP_ТАТА _Comparator within the margin of error of the measurements and calculations. We also determined the half-life and Gibbs free energy of the complex of TBP with the АВСА9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed

Forecast of development of gestational complications in women with alimentary-constitutional obesity type

In this review, there is the analysis of domestic and foreign literature on the study of mechanism and diagnosis of complications in pregnant women with obesity

WORK EXPERIENCE OF THE MULTI-PROFILE HOSPITAL ON AUTOMATION OF EXPERT ACTIVITIES IN THE HEALTH CARE QUALITY MANAGEMENT SYSTEM

Aim. Improvement of the quality of medical services provided due to a precise organization of the health care quality management system, based on the implementation of the requirements of existing regulatory documents determining expert work and its subsequent automation.Materials and methods. The quality management system in the institution is based on an active multifunctional organizational structure able to respond quickly to the detection of defects in providing medical care through the use of computerization of all areas of its activities. The automated control system of the diagnostic and treatment process proposed by us allows moving from the formal compliance with requirements established by the current regulatory documents to full management of the quality of medical care in the institution through comprehensive automation of the structure of internal quality control of medical care that ensures monitoring of all directions of the institution with the formation of automated accounting and reporting forms.Results. The introduction into the practical work of an automated quality management system for providing medical care made it possible to reduce by 5 times the time for obtaining of operative medical information, to reduce defects caused by poor registration of medical documentation by 46%, to reduce the unjustified delay of patients in the hospital by 10%, to reduce the number of cases of repeated hospitalization , due to inadequate inpatient medical care by 11%, to minimize delays at medical sub-commission for the extension of sick leave almost to zero cases.Conclusion. The automated control system of the diagnostic and treatment process proposed by us, in contrast to the existing ones at the present time, allows us to move from formal compliance with requirements established by the current normative documents to full-fledged management of the quality of medical care in the institution through comprehensive automation of the internal quality control structure of medical care providing monitoring of all activities of the institution and achievement of the recommended regulatory documents criteria of quality of medical care

Spatial structure of phosphorus-containing heterocycles. 31. 2-dialkylamino-l,3,2-dioxaphosphorinanes with four-coordinated phosphorus

1. For 2-dialkylamino-2-thiono-1,3,2-dioxaphosphorinanes, the preferred conformation is the cross with the axial position of the thiophosphoryl group. Cis-2-diethylamino-2-thiono-4-methyl-1,3,2-dioxaphosphorinane has an analogous structure with equatorial orientation of the 4-methyl. The trans isomer exists in the form of a three-component equilibrium of two conformations of the cross and a flexible form. In the 5-spirooxethane derivatives, the difference between the energies of the two cross conformations is reduced to 0.67 kcal/mole. 2. An increase has been found in the longitudinal component of anisotropy of P=S bond polarizability in 2-dialkylamino-2-thiono-l,3,2-dioxaphosphorinanes γ(P=S) 4.08 Å3 in comparison with the corresponding thiophosphates γ(P=S) 3.36 Å3. © 1983 Plenum Publishing Corporation

Spatial structure of phosphorus-containing heterocycles. 31. 2-dialkylamino-l,3,2-dioxaphosphorinanes with four-coordinated phosphorus

1. For 2-dialkylamino-2-thiono-1,3,2-dioxaphosphorinanes, the preferred conformation is the cross with the axial position of the thiophosphoryl group. Cis-2-diethylamino-2-thiono-4-methyl-1,3,2-dioxaphosphorinane has an analogous structure with equatorial orientation of the 4-methyl. The trans isomer exists in the form of a three-component equilibrium of two conformations of the cross and a flexible form. In the 5-spirooxethane derivatives, the difference between the energies of the two cross conformations is reduced to 0.67 kcal/mole. 2. An increase has been found in the longitudinal component of anisotropy of P=S bond polarizability in 2-dialkylamino-2-thiono-l,3,2-dioxaphosphorinanes γ(P=S) 4.08 Å3 in comparison with the corresponding thiophosphates γ(P=S) 3.36 Å3. © 1983 Plenum Publishing Corporation

Spatial structure of phosphorus-containing heterocycles. 31. 2-dialkylamino-l,3,2-dioxaphosphorinanes with four-coordinated phosphorus

1. For 2-dialkylamino-2-thiono-1,3,2-dioxaphosphorinanes, the preferred conformation is the cross with the axial position of the thiophosphoryl group. Cis-2-diethylamino-2-thiono-4-methyl-1,3,2-dioxaphosphorinane has an analogous structure with equatorial orientation of the 4-methyl. The trans isomer exists in the form of a three-component equilibrium of two conformations of the cross and a flexible form. In the 5-spirooxethane derivatives, the difference between the energies of the two cross conformations is reduced to 0.67 kcal/mole. 2. An increase has been found in the longitudinal component of anisotropy of P=S bond polarizability in 2-dialkylamino-2-thiono-l,3,2-dioxaphosphorinanes γ(P=S) 4.08 Å3 in comparison with the corresponding thiophosphates γ(P=S) 3.36 Å3. © 1983 Plenum Publishing Corporation