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

• Published in: Journal of molecular biology Vol. 299; no. 4; pp. 859 - 864
• Main Authors: Pemberton, I K, Muskhelishvili, G, Travers, A A, Buckle, M
• Format: Journal Article
• Language: English
• Published: England Elsevier 16.06.2000
• Subjects: Base Composition; Base Composition - genetics; Base Pairing; Base Pairing - genetics; Biochemistry, Molecular Biology; DNA Footprinting; DNA, Bacterial; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Superhelical; DNA, Superhelical - chemistry; DNA, Superhelical - genetics; DNA, Superhelical - metabolism; DNA-Directed RNA Polymerases; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Genes, Bacterial; Half-Life; Kinetics; Lasers; Life Sciences; Mutation; Mutation - genetics; Nucleic Acid Denaturation; Nucleic Acid Denaturation - genetics; Promoter Regions (Genetics); Promoter Regions, Genetic - genetics; Protein Binding; RNA, Transfer, Tyr; RNA, Transfer, Tyr - genetics; Thermodynamics; Transcription, Genetic; Transcription, Genetic - genetics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.3780

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.