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.     

A search tool for identification and analysis of conserved sequence patterns in Saccharomyces spp. orthologous promoter

We describe a web-based resource to identify, search and analyze sequence patterns conserved in the multiple sequence alignments of orthologous promoters from closely related / distant Saccharomyces spp. The webtool interfaces with a database where conserved sequence patterns (greater than 4 bp) have been previously extracted from genome-wide promoter alignments, allowing one to carry out user-defined genome-wide searches for conserved sequences to assist in the discovery of novel promoter elements based on comparative genomics. The web-based server can be accessed at http://www2.imtech.res.in/ anand/sacch_prom_pat.html.     

Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.     

Sequence of the human CALM II calmodulin gene promoter region

A clone containing a portion of the promoter region for the human bona fide CALM II gene was isolated using a human Promoter Finder DNA Walking Kit. This promoter region contains, putatively, a GC box (common in housekeeping genes), a CRE-binding site, a TATA like box and AGGGA sequences. The latter are reported to be present in genes for Ca2+ binding genes. This human promoter region exhibits overall 85% sequence identity to the corresponding region of the rat CALM II promoter but shows no identity to the corresponding region of the human CALM I or CALM III promoters.     

On the nature of human housekeeping genes

Using a collection of expressed sequence tag (EST) data, we re-evaluated the correlation of tissue specificity with genomic structure, phyletic age, evolutionary rate and promoter architecture of human genes. We found that housekeeping genes are less compact and older than tissue-specific genes, and they evolve more slowly in terms of both coding and core promoter sequences. Housekeeping genes primarily use CpG-dependent core promoters, whereas the majority of tissue-specific genes possess neither CpG-islands nor TATA-boxes in their core promoters.