Tandem Promoters for Expression of Cnu, a Nucleoid Protein, in Escherichia coli

Abstract

In Escherichia coli, initiation of chromosomal replication occurs at the replication origin, oriC. The binding of various proteins to the replication origin regulates the initiation of chromosomal DNA synthesis. Previously, Cnu, which was also named YdgT, was identified as an oriC-binding protein. 1 Cnu shares extensive sequence similarity to the nucleoid-associated Hha/YmoA protein family. Cnu and Hha together form a complex with H-NS and the complex binds oriC. 1 Cnu interacts with H-NS and StpA (H-NS paralogue) and either Cnu-StpA or Hha-StpA interaction can protect StpA from Lon-mediated proteolysis. 2 As both the cnu and hha mutation lead to the reduction of origin content, Cnu is supposed to participate in the regulation of the replication initiation. 1 Considering that replication initiation is coupled to growth rate, cnu expression should be regulated in accordance to growth stages. However, it has not known yet how cnu expression is regulated. In an effort to address this question, we examined promoter regions responsible for cnu transcription. For this purpose, we used primer extension analysis to determine 5 0 ends of cnu mRNA. Total RNAs prepared from exponentially growing or stationary-phase E. coli cells were used for primer extension analysis ( Figure 1). We found six major primer extension products whose 5 0 ends correspond to −59/−60, −96, −118/−119, −136, −143, and − 184 relative to the ATG start codon of Cnu. A homology search of the upstream sequences disclosed three promoter regions P1, P2, and P3, whose predicted transcription start sites well match to −59/−60, −136, and − 184, respectively. Since the other primer extension products to positions −96, −118/−119, and − 143 were not at the predicted transcription start sites, they might be produced from degradation products of the upstream transcripts. Therefore, the primer extension analysis suggests that cnu transcription occurs at the three tandem promoters. P3 transcription was prominent in the exponential phase, while P1 was active in the stationary phase. P2 transcription is functional in both the exponential and stationary phases.We then examined further in detail how cnu expression is regulated during growth. Total RNAs were prepared from cell cultures at different growth stages and subjected to primer extension analysis ( Figure 2). P3 was highly active in the mid-exponential phase (5 h growth), but its (a) (b) (c) Figure 1. Determination of 5 0 ends of cnu mRNA. (a) Identification of primer extension products. JM109 cells containing plasmid pHL355-36 were used for the preparation of total cellular RNAs. The 32 P-labeled primer Ext was used for primer extension. The products were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The sequencing ladders (G, A, T, and C) were prepared with primer Ext. The numbers on the left side of the gel indicate the 5 0 end positions. E and S stand for exponentially growing and stationary-phase cells, respectively. (B) Schematic map of the cnu gene and its tandem promoters. (C) Sequences o...