| Abstract |
To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage λP_R promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant P_R promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37℃, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant k_a and τ_int, as determined from the τ-plot analysis, were (6.0±0.4)×10^6 M^-1 sec^-1 and 11±5 sec, respectively. At 18℃, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. K_a for the wild-type promoter was (2.2±0.1)×10^6 M^-1 sec^-1 and τ_int was 76±5 sec. Mutant I and II exhibited differences mainly in the rate of isomerization (τ_int,I = 91±10 sec, int,II = 34±7 sec), whereas the second order rate constant k_a was similar to the wild type value. This result implies that at 18℃, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted. |
