The G+C-rich discriminator region of the tyrT promoter antagonises the formation of stable preinitiation complexes

RNA polymerase forms a highly stable preinitiation complex at many prokaryo tic promoters in the absence of ribonucleotides. These are often characteri sed by the longevity of the DNA strand-separated (open) complex in the pres ence of heparin. In contrast, such complexes are notoriously unstable at th e promoters for rRNA and tRNA under similar conditions. The high G + C cont ent within the DNA-melting region of these promoters has been implicated in this seemingly anomalous behaviour. Here, we used rapid-pulse UV laser pho to-footprinting to monitor the transient structural intermediates formed at the Escherichia coli tyrT promoter. Promoter derivatives with A+T for G/C base substitutions within the G+C-rich discriminator region (-7 to -1) augm ented the stability of complexes on both linear fragments and supercoiled p lasmid DNA. Analysis of the lifetime of the preinitiation complexes as a fu nction of the discriminator sequence reveals a direct relationship between the A + T content of the DNA-melting region and the stability of the ensuin g complex. Our results are consistent with the premise that a G/C block to DNA-untwisting and/or DNA-melting operates to prevent the formation of the stable isomers that are implicated in most other transcription initiation p athways. (C) 2000 Academic Press.