Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters

Arabidopsis thaliana carries three functional copies of the chlorophyll a/b-binding protein (cab) gene which code for an identical mature protein. DNA sequence comparison of all three cab promoters indicated that cab2 and cab3 are more closely related compared to cab1. Although the highest degree of homology was found between the TATA box and -256 of cab3 promoter, suggesting that this region plays a major role in promoter function, this promoter regions are only 47% homologous. To study whether these promoters are regulated by identical cis-acting regulatory elements, the promoters were mutated by progressive deletions and the effects on the promoter activity were measured in either transformed plants or cultured cells. It was found that the minimum sequence necessary for the light-dependent tissue-specific promoter activity of the cab3 is the 89 bp DNA fragment (between -74 and -164) at the region of the TATA and the CCAAT boxes. However, an additional 45 bp DNA fragment (between -164 and -209) upstream of the CCAAT box was necessary for the full promoter activity in the leaves. The regulatory element in the 45 bp region appears to be a positive regulator or enhancer which is specific to photosynthetic cells, since the region did not enhance the promoter activity in cultured cells. This region contains an octamer, TGCCACGT (cab2) or TGCCACAT (cab3), which is similar to the previously identified element, TGACACGT from Arabidopsis cab1 promoter. The upstream regions of the cab promoters appear to contain additional elements which are functionally distinct in each promoter since the upstream region of cab1 activated a non-functional nos promoter whereas that of cab3 did not.     

The fission yeast TFIIB-related factor limits RNA polymerase III to a TATA-dependent pathway of TBP recruitment

The RNA polymerase (pol) III-transcribed (e.g. tRNA and 5S rRNA) genes of traditionally studied organisms rely on gene-internal promoters that precisely position the initiation factor, TFIIIB, on the upstream promoter-less DNA. This is accomplished by the ability of the TFIIIB subunit, TFIIB-related factor (Brf1), to make stable protein-protein interactions with TATA-binding protein (TBP) and place it on the promoter-less upstream DNA. Unlike traditional model organisms, Schizosaccharomyces pombe tRNA and 5S rRNA genes contain upstream TATA promoters that are required to program functional pol III initiation complexes. In this study we demonstrate that S.pombe (Sp)Brf does not form stable interactions with TBP in the absence of DNA using approaches that do reveal stable association of TBP and S.cerevisiae (Sc)Brf1. Gel mobility analyses demonstrate that a TBP-TATA DNA complex can recruit SpBrf to a Pol III promoter. Consistent with this, overproduction of SpBrf in S.pombe increases the expression of a TATA-dependent, but not a TATA-less, suppressor tRNA gene. Since previous whole genome analysis also revealed TATA elements upstream of tRNA genes in Arabidopsis, this pathway may be more widespread than appreciated previously.     

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.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

Regulation of promoter activity through electrostatic interactions with RNA polymerase

Electrostatic potential profiles were calculated for σ⁷⁰-specific phage T4 promoters and four consensus-like synthetic promoters. Promoters could be classified according to the presence of specific elements of the electrostatic profile in the farther upstream region (?60 to ?100). A correlation was found between the kind of such specific element and the functional behavior of the promoter. A general scheme is proposed whereby the promoter activity is regulated through electrostatic interactions between the upstream region of promoter DNA and the RNA polymerase α-subunit.