Structural requirements for the interdomain linker of alpha subunit of Escherichia coli RNA polymerase.
The carboxy-terminal domain of the alpha subunit of Escherichia coli RNA polymerase, which is connected with the core part of RNA polymerase through a long flexible linker, plays decisive roles in transcription activation by directly 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 alpha subunit with an altered interdomain linker. Deletion of three amino acids from the linker exhibited 50% inhibition of cAMP receptor protein- (CRP-) dependent lac P1 transcription. Deletion of six amino acids completely knocked out the activity. Insertion of three amino acids did not affect the activity, whereas 40-60% inhibition was observed after insertion of one, two, or four amino acids. Substitution of 10 consecutive glycine residues resulted in nearly 90% reduction of the CRP-dependent activity, whereas 50% activity was retained after substitution of 10 proline residues or a sequence expected to form a strong alpha-helix. Essentially the same results were obtained with UP element-dependent rrnB P1 transcription. These observations altogether suggest that (i) sufficient length of the interdomain linker is required for transcription activation mediated by the alpha carboxy-terminal domain, (ii) the linker is not totally unstructured but has structural and torsional preferences to facilitate positioning of the carboxy-terminal domain to a proper location for the interaction with CRP and UP element, and (iii) CRP-dependent activation and UP element-dependent activation share a common intermediary state in which the positioning of the alpha carboxy-terminal domain is of primary importance.
Fujita
N
N
Department of Molecular Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan. nfujita@lab.nig.ac.jp
Endo
S
S
Ishihama
A
A
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D012407:rRNA Operon
D020121:5' Untranslated Regions; D002373:Cyclic AMP Receptor Protein; D010446:Peptide Fragments; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit