Mapping of T7 RNA polymerase active site with novel reagents - oligonucleotides with reactive dialdehyde groups

DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction- modification enzymes.

To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose o...

PROBING THE ACTIVE SITE OF RHODANESE WITH DISULFIDE REAGENTS

RNA-binding site in T7 RNA polymerase.

Recent models of RNA polymerase transcription complexes have invoked the idea that enzyme-nascent RNA contacts contribute to the stability of the complexes. Although much progress on this topic has been made with the multisubunit Escherichia coli RNA polymerase, there is a paucity of information regarding the structure of single-subunit phage RNA polymerase transcription complexes. Here, we pho...

Mapping the active site of yeast RNA polymerase B (II).

Yeast RNA polymerase B (II) was incubated with a collection of 13 different nucleotide derivatives and affinity labeled by allowing DNA-directed phosphodiester bond formation. The 32P-labeled site was localized in the C-terminal part of the B150 subunit by microsequencing a proteolytic fragment, then further mapped by a combination of extensive or single-hit chemical cleavage reactions and anal...

M13 vectors with T7 polymerase promoters: transcription limited by oligonucleotides.

Synthetic sequences corresponding to T7 promoters have been cloned into the vectors H13mplO and M13mpl8 at the EcoRl site and into M13mpll and M13mpl9 at the Hlndlll site, creating mICE 10,11,18 and 19. These sequences of 33-base pairs include the region essential for activity of T7 promoters (1). The inserts preserve the g-galactosldase cloning marker and restore the initial unique restriction...