INTRAMOLECULAR TAT TRIPLEX IN (DA)(58).(DT)(58) - INFLUENCE OF IONS

Supercoil-stabilized intramolecular triplexes have been described unde r various conditions in different polypurine polypyrimidine sequences such as (dG)(n),(dC)(n) and mixed sequences including d(GA)(n).d(CT)(n ) while information about the triplexes in (dA)(n).(dT)(n) is scarce. Using osmium tetroxide complexes and diethyl pyrocarbonate as structur al probes, we show a pyrimidine.purine.pyrimidine (TAT) tripler in (dA )(58).(dT)(58) sequence in a supercoiled plasmid pE19. Strong modifica tion of approximately six central thymines and approximately six T's a t the 3'-end of the (dT)(58) stretch as well as the DEPC modification of the 5'-haif of the (dA),, strand suggested the prevalence of the H- y3 tripler conformer. At native superhelix density, optimum conditions for the tripler formation were close to 1 mM MgCl2, pH 8.5. At room t emperature and MgCl2 concentrations below 0.5 and above 5 mM, almost n o tripler was formed. It is suggested that the absence of the tripler at higher MgCl2 concentrations is due to the stabilization of the dupl ex by Mg2+ ions which prevents the duplex opening necessary for the tr ipler formation. At higher temperatures, favorable for duplex opening (e.g. 55 degrees C), the TAT tripler is formed even in the presence of 10 mM MgCl2. Among Ca2+ Sr2+, Ba2+, Cd2+, Zn2+ and Ni2+, only Ca2+ an d Sr2+ yielded a modification pattern similar to that obtained with Mg 2+; the modification pattern produced in the presence of Sr2+ was, how ever, much less intense. In the presence of 1 mM MgCl2 a decrease in p H from 8.5 to 7.7 resulted in a strong decrease of the tripler content . At highly negative superhelix density, the conditions for tripler fo rmation were less stringent, and the tripler was observed even in the absence of MgCl2.