Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attentuation
Poulsen, P.; Bonekamp, F.; Jensen, K.F.
EMBO Journal 3(8): 1783-1790
1984
ISSN/ISBN: 0261-4189 PMID: 6207018 Document Number: 223871
Protein synthesis in minicells showed that the DNA immediately preceding the pyrE gene of E. coli directs the formation of considerable amounts of a polypeptide (MW .apprx. 30,000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream from the start site of pyrE, just prior to the GC-rich symmetry region of a sequence with features characteristic of a rho-independent transcription terminator. Deletion of the start of this 238 codon-long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that genes ORF and pyrE constitute an operon. S1 nuclease digestion of RNA-DNA hybrids revealed that both genes are transcribed from 2 promoters (P1 and P2) located in front of the ORF start. When the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. Evidently, pyrE expression is controlled by a UTP modulated intercistronic attentuation.