Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 299, Issue 4, Pages 859-864Publisher
ACADEMIC PRESS LTD
DOI: 10.1006/jmbi.2000.3780
Keywords
preinitiation complex; RNA polymerase; stable RNA; discriminator; UV laser photo-footprint
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RNA polymerase forms a highly stable preinitiation complex at many prokaryotic promoters in the absence of ribonucleotides. These are often characterised by the longevity of the DNA strand-separated (open) complex in the presence of heparin. In contrast, such complexes are notoriously unstable at the promoters for rRNA and tRNA under similar conditions. The high G + C content within the DNA-melting region of these promoters has been implicated in this seemingly anomalous behaviour. Here, we used rapid-pulse UV laser photo-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) augmented the stability of complexes on both linear fragments and supercoiled plasmid DNA. Analysis of the lifetime of the preinitiation complexes as a function of the discriminator sequence reveals a direct relationship between the A + T content of the DNA-melting region and the stability of the ensuing 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 pathways. (C) 2000 Academic Press.
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