Structural requirements for the interdomain linker of alpha subunit of Escherichia coli RNA polymerase

The carboxy-terminal domain of the a subunit of Escherichia coli RNA polyme rase, which is connected with the core part of RNA polymerase through a lon g flexible linker, plays decisive roles in transcription activation by dire ctly interacting with a large number of transcription factors and upstream (UP) element DNA. Here we constructed a set of mutant RNA polymerases, each containing a mutant a subunit with an altered interdomain linker. Deletion of three amino acids from the linker exhibited 50% inhibition of cAMP rece ptor protein- (CRP-) dependent lac P1 transcription. Deletion of six amino acids completely knocked out the activity. Insertion of three amino acids d id not affect the activity, whereas 40-60% inhibition was observed after in sertion of one, two, or four amino acids. Substitution of 10 consecutive gl ycine residues resulted in nearly 90% reduction of the CRP-dependent activi ty, whereas 50% activity was retained after substitution of 10 proline resi dues or a sequence expected to form a strong alpha-helix. Essentially the s ame results were obtained with UP element-dependent rrnB P1 transcription. These observations altogether suggest that (i) sufficient length of the int erdomain Linker is required for transcription activation mediated by the a carboxy-terminal domain, (ii) the linker is not totally unstructured but ha s structural and torsional preferences to facilitate positioning of the car boxy-terminal domain to a proper location for the interaction with CRP and UP element, and (iii) CRP-dependent activation and UP element-dependent act ivation share a common intermediary state in which the positioning of the a lpha carboxyterminal domain is of primary importance.