E. coli promoter spacer regions contain nonrandom sequences which correlate to spacer length.

The -10 and -35 regions of E. coli promoter sequences are separated by a spacer region which has a consensus length of 17 base-pairs. This region is thought to contribute to promoter function by correctly positioning the two conserved regions. We have performed a statistical evaluation of 224 spacer sequences and found that spacers which deviate from the 17 base-pair consensus length have nonrandom sequences in their upstream ends. Spacer regions which are shorter than 17 base-pairs in length have a significantly higher than expected frequency of purine-purine and pyrimidine-pyrimidine homo-dinucleotides at the six upstream positions. Spacer regions which are longer than 17 base-pairs in length have a significantly higher than expected frequency of purine-pyrimidine and pyrimidine-purine hetero-dinucleotides at these positions. This suggests that the nature of the purine-pyrimidine sequence at the upstream end of spacer regions affect promoter function in a manner which is related to the spacer length. We examine the spacer sequences as a function of spacer length and discuss some possible explanations for the observed relationship between sequence and length.     

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.     

Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA.

Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure. The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dinucleotide at position -15, -14. In addition, several weakly conserved A and T residues are present upstream of the -35 region. Analysis of dinucleotide composition reveals A2- and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: An tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions. When compared with larger regions of the genome, upstream promoter regions have an excess of An and Tn sequences for n > 4. These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70. This sequence conservation is discussed in light of recent evidence that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.     

The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.     

Adenovirus E3-early promoter: sequences required for activation by E1A.

To identify sequences within the adenovirus-5 E3 promoter necessary for E1A trans-activation, a series of promoter deletion mutants were constructed and analysed. A region between positions -82 and -105 was shown to be critical both for E1A induced expression as well as uninduced expression. The importance of this region was confirmed by constructing hybrid promoters consisting of E3 and Herpes simplex virus thymidine kinase sequences. The E1A insensitive tk promoter could be converted to an E1A sensitive promoter by replacing sequences upstream of position -79 with the corresponding region of the E3 promoter. This critical region of the E3 promoter contains a sequence 5' AGATGACTA3' which is also present in important upstream regions of the E2A and E4 promoters.     

Effect of DNA structural flexibility on promoter strength--molecular dynamics studies of E. coli promoter sequences

To study possible correlations between promoter activity and the structural flexibility of the DNA helix, we have carried out unrestrained molecular dynamics simulations of the -10 consensus region sequence and five variants forming the -10 region of various Escherichia coli promoter sequences. Analyses of the trajectories obtained from the simulations show that the consensus sequence has a pattern of two structurally flexible nucleotide steps sandwiched between two stiff steps. In the other sequences, this pattern varies in consonance with the change in the sequence. The variations in the patterns show correlation with the promoter strength.     

Up-promoter mutations in the lpp gene of Escherichia coli.

The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the -10 and the -35 regions of the lpp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The beta-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(P1) and TATAAT (consensus sequence; P2), respectively. Similarly, it increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(R1) and TTGACA (consensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result in the highest activity. With use of the improved lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.