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.     

Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression

The isolation and characterization of fruit-specific promoters are critical for the manipulation of the nutritional value and quality of fruits by genetic engineering. The analysis of regulatory sequences of many ripening-related genes has remained elusive for many species due to their low transformation efficiency and/or lengthy regeneration of a small number of transgenic plants. Strawberry is an important crop and represents one of the most widely studied non-climacteric model systems. However, until recently, its difficult regeneration has limited the functional study of promoters by stable transformation. A protocol based on biolistic transient transformation has been developed in order to study the function of promoters in a fast and efficient manner in strawberry fruits. The protocol has been applied to the study of the GalUR promoter, a gene involved in the biosynthesis of vitamin C in this fruit. The activity of the GalUR promoter is restricted to the fruit, being strictly dependent on light. The analysis of deletion series revealed the presence of a minimum activation region 397 bp upstream of the gene with a putative G-box motif, and a negative regulatory region between -397 and -518 bp, where an I-box was identified. The transient assay has been used to study the activity of the tomato polygalacturonase and the pepper fibrillin promoters in strawberry fruits. Whereas slight activity was observed with the fibrillin promoter, no significant activity was found with the polygalacturonase promoter. The GalUR promoter in transiently transformed ripe tomato fruits showed no activity, indicating the presence of regulatory sequences specific for its function in strawberry fruit.     

Consensus methods for finding and ranking DNA binding sites. Application to Escherichia coli promoters

There have been many different approaches employed to define the "consensus" sequence of various DNA binding sites and to use the definition obtained to locate and rank members of a given sequence family. The analysis presented here enlists two of these approaches, each in modified form, to develop a highly efficient search protocol for Escherichia coli promoters and to provide a relative ranking of these sites showing good agreement with in vitro measurements of promoter strength. Schneider et al. have applied Shannon's index of information content to evaluate the significance of each position within the consensus of a family of aligned sequences. In a formal sense, this index is only applicable to a group of sequences, providing at each position a negative entropy value between zero (random) and two bits (total conservation of a single base) for sequences in which all bases are equally represented. A method for evaluating how well an individual sequence conforms to the information content pattern of the consensus is described. A function is derived, by analogy to the information content of the sequence family, for application to individual sequences. Since this function is a measure of conformity, it can be used in a search protocol to identify new members of the family represented by the consensus. A protocol for locating E. coli promoters is presented. The Berg-von Hippel statistical-mechanical function is also tested in a similar application. While the information content function provides a superior search protocol, the Berg-von Hippel function, when scaled at each position by the information content, does well at ranking promoters according to their strength as measured in vitro.     

Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites

Twenty-nine early promoters from bacteriophage T4 and 14 early promoters from bacteriophage T6 were isolated using vector M13HDL17, a promoterless derivative of M13mp8 carrying a linker sequence, the bacteriophage lambda-terminator tR1, and the lacZ' gene including part of its ribosome-binding site. The consensus sequence for the T4 promoters is: (sequence; see text). Ribosome-binding sites of T4 share the sequence: 5'...g.GGAga..aA.ATGAa.a...3' The consensus sequence of the T4 early promoter regions is significantly different in sequence and length from that of Escherichia coli promoters. Only one of the promoters detected with vector M13HDL17 resembled a typical bacterial promoter. The high information content raises the possibility that additional proteins recognize and contact nucleotides within the promoter region. All T4 early promoters also carry DNA sequences that could support DNA curving, a structural feature that might contribute to promoter recognition.     

The signal for growth rate control and stringent sensitivity in E. coli is not restricted to a particular sequence motif within the promoter region.

Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region. The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant. The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene. Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response. Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter. A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation. The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control. However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.     

Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.     

Compilation and analysis of Escherichia coli promoter DNA sequences.

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.