Book of Abstracts: Albany 2003
June 17-21 2003
Temperature-induced Alterations Within E.coli RNA Polymerase α-subunit Contact Region with T7D Promoter Affect the Mode of Transcription Initiation
Using fluorescent probe covalently attached to cys-269 of RNA polymerase (RP) α-subunit a set of conformation transitions has been previously revealed upon complex formation with T7D. Besides alterations reflecting association with DNA, close and open promoter complexes formation, this set includes one additional transition observed above 30°C (1). Taken into account the location of the label, these data imply conformational rearrangements near one or both α-subunits contact modules (-52/-48 and/or -42/-38 relative to the transcription start site (2)). To test this possibility RP?T7D complexes were analyzed by DNase I and monoacetyl-4-hydroxy-aminoquinoline 1-oxide (AcHAQO) footprinting techniques at 22 and 37°C. Temperature dependence against DNAse exhibited at least 2 bonds on the bottom strand (-36/-35; -48/-47) and one on the top strand (-43/-42). AcHAQO probing revealed abnormal digestion of A (-35) and A (-34) on the bottom strand at 22°C and better protection of G (-43, -39, -38) on the top strand at 37°C. Thus temperature variations really induce well-pronounced structural changes in the α contact regions. To test functional significance of these transitions, the rate of abortive and productive RNA synthesis has been estimated by using fluorescent analog of UTP, UTP-γ-ANS. Accumulation of RNA-products either in the course of abortive (only 2 initiating substrates added) or productive (all NTPs added) reaction was quantified on the basis of increased emission intensity of ANS detached with pyrophosphate. Rate/temperature plots revealed unexpected increase in the slope of the curve for abortive reaction and decrease in the slope of the curve for productive elongation above ~30°C, thus indicating heat-induced changes in the equilibrium between abortive and productive complexes. To test the nature of abortive complexes (transient or "moribund" (3)) the time-course of single-round transcription has been monitored at 21, 30 and 37°C. RNA products were separated by Urea-PAAG electrophoresis. Besides transcripts synthesized in the course of productive reaction essential amount of abortive products have been registered. The later products were accumulated for at least 30 min and their yield increased with temperature rise, thus indicating that some abortive RP-T7D complexes are not able to carry out productive synthesis and increase of temperature stabilizes them. Efficiency of productive elongation from T7D may therefore be regulated by thermal variation, which modifies promoter contacts with α. Importance of these contacts for elongating complex formation has been also discussed for T7A1 (4) and may have general significance.
Supported by Russian Foundation for Basic Research (03-04-48339, 01-04-97006).
V. V. Chasov*
Institute of Cell Biophysics RAS