The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein

Abstract

DNase I protection experiments have indicated that the cyclic AMP-catabolite gene activator protein complex binds to two regions preceding the chloramphenicol acetyl transferase (cat) gene in Escherichia coli. One of these lies adjacent to the RNA polymerase binding site, whereas the second lies approximately 130 base pairs upstream from the starting point of transcription. Additional DNase protection experiments and in vitro transcription experiments with modified templates indicate that the catabolite gene activator protein site proximal to the cat promoter functions independently of the distal site, indicating that in vitro the second of these sites is not required for transcriptional activation of the cat gene.In Escherichia .coli, the cyclic AMP (cAMP)catabolite gene activator protein (CAP) complex acts as a regulator of certain genes or operons, the most notable being the sugar utilization operons, although several other systems have been shown to be under a similar mode of regulation (20). Early studies with the lac operon suggested that the cAMP-CAP complex might recognize certain sequences in the vicinity of the RNA polymerase binding site (5), and Gilbert (7) has suggested that transcriptional activation could occur by (i) direct interaction between CAP and RNA polymerase or (ii) cAMP-CAPinduced destabilization of DNA. At present there is no direct evidence to suggest which of these mechanisms prevails, and in fact it may be that both are required. Ebright and Wong (6) have presented strong evidence that direct intercalation between the adenyl moeity of cAMP and a thymine residue within the CAP binding site may destabilize the DNA sufficiently to allow enhanced RNA polymerase interaction. Additionally, chemical and enzymatic probes have indicated that CAP and RNA polymerase occupy adjacent sites on the promoter, as in the lac promoter (26), or overlapping sites, as in the araC (10, 18), gal (27), pBR-P4 (21), and ompA (16) promoters, where contact between CAP and RNA polymerase could occur. The promoter for the araBAD operon has a CAP site displaced from the RNA polymerase site and contains an t Present address:

Action of CAP on the malT promoter in vitro

“…The size of the CAP binding site, approximately 26 bp according to the DNase I footprinting data, is similar to that found at other promoters ( Fig. 6A) (21,22,31,34,40,42,45,46). Several authors have noted the existence of sequence homologies between the different CAP binding sites (13,32,34,45).…”

Tandem CRP binding sites in the deo operon of Escherichia coli K-12.

“…Since the levels of cAMP required to induce different genes controlled by cAMP/CRP are not identical, it might be expected that only part of the DNA sequence of different CRP binding sites is conserved. In accordance with this prediction, the CRP target has been found to vary for several genes, not only with respect to the DNA sequences and symmetry characters but also with respect to the distance between the binding site and the start of transcription (Reznikoff and Abelson, 1978;Simpson 1980;Taniguchi et al, 1979;Ogden et al, 1980;Lee et al, 1981;Queen and Rosenberg, 1981;Le Grice et al, 1982). In lac, CRP binds between -74 and -50; in ara between -106 and -82; and in gal, cat and in promoter P4 on the plasmid pBR322 it binds around -40. Thus, cAMP/CRP is able to activate transcription from at least three different locations.…”

“…The two conserved regions are arranged around a center of symmetry ( Figure 5) -suggesting that the symmetric elements may correspond to symmetric features of the CRP dimer. Also the CRP target of the ompA promoter shows this sequence homology (Movva et al, 1981), whereas the right hand site of the cat target contains four changes from the consensus (Le Grice et al, 1982). Each CRP-binding site includes (on one side) the consensus sequence 5' -AANT-GTGANNTNNNNCA-3' (Ebright, 1982), or a sequence differing by two bases, plus (on the other side) a related but more variable sequence, differing by two to five bases.…”

“…In vitro data on gene fusions as well as homology with other known CRP sites, both with respect to DNA sequence and position, strongly indicate that both CRP target sites are needed for high expression from the P-2 promoter. The presence of two distinct CRP targets in the promoter region of lac (Schmitz, 1981), ara (Lee et al, 1981), and cat (Le Grice et al, 1982) has been described. However, both in ara and lac, one of the sites is a non functional low-affinity target and in vitro data suggest that the two CRP targets in cat regulate two distinct promoters.…”

Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria.

“…DNA sequence analysis of the type I CAT gene and DNase protection experiments identified two CAP sites in the promoter-proximal region of the gene (227). One site is located 130 bp upstream from the start point of transcription but is not required for the stimulation of transcription.…”