Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.     

Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.     

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.     

Gene Expression in Bacteria Directed by Plant-specific Regulatory Sequences

The regulation of gene expression represents a specific process which has different structural and functional requirements in different groups of organisms. It is thus assumed that regulatory sequences of eucaryotes cannot be recognized in procaryotes. This assumption is of interest for risk assessments of the environmental impact of deliberate release experiments with genetically modified organisms. In order to analyse the extent of heterologous gene expression caused by the transfer of plant-specific regulatory sequences into bacteria, we constructed fusions between plant-specific regulatory sequences and the coding regions of the luxAB genes for the luciferase of the bioluminescent bacterium Vibrio harveyi, transferred the fusions into different bacterial species and measured the luminescence to quantify the expression of the luciferase genes. The regulatory sequences investigated included (a) the 35S promoter of the Cauliflower mosaic virus, (b) the B33 promoter of a class I patatin gene of potatoes, (c) the promoter of the ST-LS1 gene of potatoes and (d) the promoter of the rolC gene of Agrobacterium rhizogenes. We could show that in addition to the 35S promoter, which has already been described as being recognized in Escherichia coli, the sequences containing the B33 and the ST-LS1 promoters are recognized in bacteria. Luciferase gene expression promoted by the sequence with the ST-LS1 promoter could be observed in E. coli, Yersinia enterocolitica and Agrobacterium tumefaciens. Comparison of the luminescence caused by fusions between luxAB and different promoters on the chromosome and on an endogenous plasmid of Y. enterocolitica demonstrated that the level of the heterologous gene expression caused by the fragment with the ST-LS1 promoter was within the range of gene expression levels caused by endogenous promoters of Y. enterocolitica.     

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the -10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A37 for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A37-RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to -10 sequences, DNA sequence at -35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in -10 and -35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions.