Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Structure of the promoter region for the rrnB gene in Escherichia coli.

The nucleotide sequence of the promoter region for the rrnB gene of E. coli had been determined by the Maxam-Gilbert technique. The 700 bp long sequence had been compared with the published sequences of four other rRNA promoter regions. The rrnB sequence was found to be homologous with the rrnA promoter sequence till the 370th base upstream from the coding region of mature 16S rRNA. The significance of this homology is discussed and a tentative model is proposed to account for the unusual properties of the rRNA promoters.     

A common structural feature in promoter sequences of E. coli.

We have searched promoter regions of E. coli, structural genes of the same organism, and computer-generated random sequence DNA for the occurrence of common structural features. This is done by converting the base sequence to a series of numbers representing the sequence of helix twist angles and examining these numerical sequences statistically. Common structural features are shared by the promoter regions with a much higher frequency than are found in structural genes or in random sequences. These structures appear to be scattered randomly throughout the promoters, both in terms of the number of such structures per promoter and in terms of location within each promoter. One particular structure consisting of five successive helix twist angles is reported, along with a list of 60 different hexanucleotide sequences that share this structure. The locations of these structural elements in 61 E. coli promoters are also tabulated.     

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.     

Training back-propagation neural networks to define and detect DNA-binding sites

A three layered back-propagation neural network was trained to recognize E. coli promoters of the 17 base spacing class. To this end, the network was presented with 39 promoter sequences and derivatives of those sequences as positive inputs; 60% A + T random sequences and sequences containing 2 promoter-down point mutations were used as negative inputs. The entire promoter sequence of 58 bases, approximately -50 to +8, was entered as input. The network was asked to associate an output of 1.0 with promoter sequence input and 0.0 with non-promoter input. Generally, after 100,000 input cycles, the network was virtually perfect in classifying the training set. A trained network was about 80% effective in recognizing 'new' promoters which were not in the training set, with a false positive rate below 0.1%. Network searches on pBR322 and on the lambda genome were also performed. Overall the results were somewhat better than the best rule-based procedures. The trained network can be analyzed both for its choice of base and relative weighting, positive and negative, at each position of the sequence. This method, which requires only appropriate input/output training pairs, can be used to define and search for any DNA regulatory sequence for which there are sufficient exemplars.     

Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.     

Conserved features of coordinately regulated E. coli promoters

E. coli promoters which are coordinately regulated in response to amino acid limitation contain conserved nucleotide sequences immediately 3' to -10 region. These sequences contain predominantly either GC or AT residues depending on whether the response is respectively negative or positive. Certain classes of promoters also contain conserved sequences upstream of the primary promoter. In tRNA genes these sequences could act as a secondary polymerase binding site.