A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase–substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase–substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase–substrate complexes that are suitable for future applications characterizing cell signaling pathways.     

NusA changes the conformation of Escherichia coli RNA polymerase at the binding site for the 3' end of the nascent RNA.

A conformational change in Escherichia coli RNA polymerase induced by NusA was detected by utilizing photocrosslinking. A change in the binding site for the 3' end of the RNA occurred, and NusA increased interactions of the RNA with the beta subunit of the polymerase. NusA was not contacted by the 3' end of the RNA.     

The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli.

Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA. These reactions were carried out by E. coli RNA polymerase on T7 DNA in the presence of rifampicin. The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined. On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds. The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not.