Abstract
The bacterial RNA polymerase holoenzyme containing the final sigma(54) subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the final sigma(54) DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the final sigma and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by final sigma(54), and holoenzyme and is involved in activator-dependent final sigma isomerization. As part of the final sigma(54)-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the -12 promoter region to set basal and activated levels of transcription.
Highlights
factors play a pivotal role in bacterial transcription
Single amino acid substitutions were obtained that led to defects in promoter binding, isomerization, and activator-dependent transcription
Mutations in 54—Enzyme and chemical cleavage of 54 suggested that the patch of 54 that can be UV-cross-linked to promoter DNA resided between or included residues Trp-328 to Cys-346 [14, 17]
Summary
factors play a pivotal role in bacterial transcription. Through their association with core RNA polymerase they function in promoter-specific initiation. We have shown that the invariant residue Arg-336, within the cross-linking patch, is important for maintaining the holoenzyme as a closed complex, silent for transcription before activation [22]. Region I binds core polymerase, and deletion of Region I alters the conformation of the carboxyl-terminal DNA binding domain of 54 as part of the holoenzyme, suggesting a linked interaction between amino and carboxyl parts of 54 [13, 24]. Identification of 54 sequences involved in interaction with promoter DNA provides a basis for understanding the mechanism of transcription initiation by enhancer binding activators. We deleted the adjacent surface-exposed element, amino acids 310 –328, which links the cross-linking patch with a domain that enhances DNA binding [20, 21].
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