Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion

Abstract

The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990 starved cells. The protein extract from a thymine-starved culture protected several sites in the promoter region from DNase I digestion (47). Here, we report the identity of this protein as the E. coli factor for inversion stimulation (Fis).Fis is an 11.2-kDa protein initially identified by its ability to stimulate Hin-, Gin-, and Cin-mediated DNA inversion (18,23,25). The involvement of Fis in other site-specific recombinations, including excision and integration recombination reactions of bacteriophage X DNA, was also demonstrated (3,4,44). Fis has also been shown to be a transcriptional activator at rRNA and tRNA promoters in E. coli (6,38). In vitro footprinting experiments show protection of specific sites in the E. coli oriC sequence by Fis (17), and examination of DNA synthesis in fis gyrB double mutants indicates a synergistic involvement of Fis and DNA gyrase in the initiation of DNA replication at oriC (14).The fis gene has been cloned, sequenced, and mapped to 72 min on the E. coli chromosome (23,26). The gene encodes an 11,239-Da, 98-amino-acid polypeptide. The fis operon promoter has been located 1 kb upstream of the fis start codon (36), and Fis synthesis is autoregulated. Transcription of fis is controlled as part of the stringent response. A degenerate consensus binding site for Fis has been proposed: (G/T) nnYRnn(A/T)nnYRnn(C/A) (where Y is a pyrimidine, R is a purine, and n is any nucleotide) (22). However, local DNA topology and the ability of the DNA duplex to bend, accommodating the helix-turn-helix DNA-binding domains of the Fis dimer, appear to be more important in determining Fis binding at any given site (51) than the sequence per se. In activating transcription of stable RNA operons in E. coli, binding of Fis to upstream activating sequences lowers the dissociation constant of the RNA polymerase-promoter complex (34).

Expression Analysis of the nrdHIEF Operon fromEscherichia coli

“…Many genes having a growth phaseand growth medium-dependent regulation are under global regulatory mechanisms like those mediated by RpoS, Fis, or the intracellular levels of cAMP (35,36). Our data indicate, however, that these global regulators are not responsible for the nrdHIEF expression pattern, thus making a difference with the nrdAB operon, which is known to be under the positive regulation of Fis protein (37).…”

Ribonucleotide Reductases

“…In general, transcription is increased when DNA replication is inhibited or when conditional DNA mutants are shifted to nonpermissive conditions (104). DnaA and Fis proteins bind to specific promoter sites and activate transcription but do not couple transcription to the cell cycle (105,106). Instead, a cis-acting ATP-rich region located downstream of the Fis footprint area and preceding an inverted repeat is important for coupling nrdAB expression to the cell cycle and also for increasing transcription after inhibition of DNA synthesis (107).…”

A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA

“…Increased transcription of nrdEF, which encodes nucleotide reductase needed for deoxyribonucleotide biosynthesis, is a common response to DNA damage and replication-fork arrest in several organisms (50,51). The increase in ribonucleotide reductase in Saccharomyces cerevisiae contributes to survival after DNA damage; higher levels of dNTPs are thought to be necessary for error-prone DNA replication (52).…”