Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization

Fifty-two of the best characterized Escherichia coli promoters in the Hawley and McClure [1983) Nucleic Acids Res. 8, 2237-2255) listing were used to determine the distribution of information content in promoters and to describe the basic features underlying the existence of several different promoter spacing classes, which are defined by the number of bases separating the -35 and -10 regions. The contact regions at -35 and -10 do not, on the average, contain sufficient information to specify a promoter. The search for additional specifying bases led to two conclusions: 1) the consensus nucleotide sequence in the noncontact regions of a promoter appears to be distinct for each of the major promoter spacing classes; 2) promoters appear to contain a 15-20 base subset of the 40-50 additional optimal noncontact bases. This improved view of the extended consensus sequence allows the detection of a 10-base degenerate palindrome which may be the basic unit of promoter structure. Contiguous direct repeats of this sequence produce a sequence closely related to the consensus for the 18-base pair spacing class. This underlying structure is also evidenced in the 17- and 16-base pair spacing classes; however, the start points of the fourth and subsequent repetitions of the sequence element are moved one and two bases upstream, respectively, relative to their location in the 18-base pair spacing class. These consensus sequences, when viewed in a helical format, all present the opportunity for two alternative sets of a dyad repeat. The -35 region is common to both sets and is paired with an extended -10 region in one set and with a pseudo-10 region in the other. Possible implications of these arrangements are discussed.     

Sequence elements determining ampC promoter strength in E. coli

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.     

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the –10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the –10 and –35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions –15 and –14. Promoter activity is greatest when the ‘non-template’ strand carries T and G at positions –15 and –14, respectively. Promoter activity can be further enhanced by a second T and G at positions –17 and –16, respectively, immediately upstream of the first ‘TG motif’. Our results show that the base sequence of the DNA segment upstream of the –10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of ‘TG motifs’ upstream of the promoters’ –10 elements. We conclude that correctly placed ‘TG motifs’ are found at over 20% of E.coli promoters.     

High level heterologous expression in E. coli using mutant forms of the lac promoter.

Single base deletions in the lac promoter which reduced the 18bp spacing between the -35 and -10 homology regions to 17bp, increased the strength of the promoter. A single base substitution (T----G) in the -35 region to generate the consensus sequence TTG-ACA increased the strength further and no longer required a 17bp spacing. The mutated lac promoter was as powerful as a shorter form of the tac promoter which lacked two AT-rich regions upstream of the -35 region, and expressed the P69 surface antigen (pertactin) of Bordetella pertussis to 30-40% total cell protein and tetanus toxin fragment C to 16-20% total cell protein.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.