Book of Abstracts: Albany 2005
Local DNA Structural Features in the Contact Region with E.coli RNA Polymerase α Subunits Affect Transcription Complex Formation
At least four structural modules in the promoter DNA are ideally required to form binary complexes with bacterial RNA polymerase. Two of them (elements -35 and -10) are recognized by the σ subunit of the enzyme forming base-specific contacts in the DNA major groove. Two others interact with two identical α subunits forming structure-specific contacts in the minor groove. It is generally accepted, that rigid polyA(T)-tracts characterized by the narrowed minor groove are an optimal targets for interaction with α subunits and many known promoters contain such motifs. The putative contact regions with α subunits are also characterized by the high frequency in the presence of flexible dinucleotide TA, which in many cases are located in the spacer separating two contacting modules. Here we report that the presence and positioning of TA may be important for promoter activity.
Promoter D of phage T7 DNA was used as a model template. The α subunits interact with -52/-48 and -43/-39 regions of this promoter (OH-radical footprinting). The spacer between them contains flexible dinucleotide TA in position -46. We found that substitution of this dinucleotide by rigid polyT-tract (TTTAA→TTTTT) induces DNA bending in this region (computer modeling of three-dimensional structure), alters the topology of α-promoter contacts (DNAse footprinting) and inhibits synthesis of T7D-RNA measured by the single round transcription assays in the presence of competing promoter, but the association constant with RNA polymerase (gel retardation experiments) and efficiency of open complex formation tested by KMnO4 probing remain unchanged. That means that flexibility in the contact region with α dimmer may be required for promoter clearance and productive elongation. One base pair shifting of dinucleotide TA to position -47 showed no any affect on the transcription efficiency but affected local structural features of the promoter DNA (DNAse I probing) decreasing the size of contact surface with the distal α and association constant with RNA polymerase. That means that the kinetics of the transcription complex formation depends on the positioning of the flexible step. Conformational flexibility in the upstream region of the promoter DNA may, therefore, be considered as an independent and significant factor balancing its binding propensity in course of RNA polymerase-promoter interaction and functional properties of transcriptional complexes under formation.
Supported by RFBR grant 03-04-48339 and RFBR-Ministry of Industry and Science(Moscow Region) (grant 04-04-97280).
E. G. Kostyanicina*
Institute of Cell Biophysics RAS