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

Conservation and revised annotation of the T reponema denticola prcB‐prcA‐prtP locus encoding the dentilisin ( CTLP ) protease complex

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97510/1/omi12013.pd

Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself