Molecular Mechanisms of Transcription Initiation-Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation.

Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the ß-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery.

Archaeal TFEα/ß is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and ß subunits. Here we have identified and characterised the function of the TFIIEß homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEß and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/ß and its engagement with the RNAP clamp. TFEα/ß stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the ß subunit and the promoter sequence. Our results suggest that archaeal TFEα/ß is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

Global analysis of the regulon of the transcriptional repressor LexA, a key component of SOS response in Mycobacterium tuberculosis.

The DNA damage response is crucial for bacterial survival. The transcriptional repressor LexA is a key component of the SOS response, the main mechanism for the regulation of DNA repair genes in many bacteria. In contrast, in mycobacteria gene induction by DNA damage is carried out by two mechanisms; a relatively small number of genes are thought to be regulated by LexA, and a larger number by an alternate, independent mechanism. In this study we have used ChIP-seq analysis to identify 25 in vivo LexA-binding sites, including nine regulating genes not previously known to be part of this regulon. Some of these binding sites were found to be internal to the predicted open reading frame of the gene they are thought to regulate; experimental analysis has confirmed that these LexA-binding sites regulate the expression of the expected genes, and transcriptional start site analysis has found that their apparent relative location is due to misannotation of these genes. We have also identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs.

Mycobacterium tuberculosis H37Rv sigC is expressed from two promoters but is not auto-regulatory.

The extracytoplasmic function (ECF) sigma factor SigC has been implicated in the pathogenesis of Mycobacterium tuberculosis but control of its expression and activity is poorly understood. No proteins that interact with SigC have been detected leading to the suggestion that this sigma factor may be primarily controlled at the level of transcription. It has been suggested that SigC may be autoregulatory and a role has also been proposed for SigF in the expression of sigC. In this study we identified two promoters that were active under standard growth conditions by a combination of transcript start site mapping and promoter-lacZ fusion assays. The dominant promoter, P1, closely resembled mycobacterial SigA-dependent promoters, and introduction of a single base change at the conserved A of the -10 region eliminated promoter activity. Although the sequence of the other, P2, closely resembled the reported SigC consensus motifs, expression directed by this promoter was unaltered in a ΔsigC mutant strain, or in strains defective in other ECF sigma factors for which some similarity in consensus sequences was apparent. Comparison of the effects of different changes in the -10 region suggested that the P2 promoter was most likely recognised by SigA.

SigG does not control gene expression in response to DNA damage in Mycobacterium tuberculosis H37Rv.

Expression of the Mycobacterium tuberculosis sigG sigma factor was induced by a variety of DNA-damaging agents, but inactivation of sigG did not affect induction of gene expression or bacterial survival under these conditions. Therefore, SigG does not control the DNA repair response of M. tuberculosis H37Rv.

Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions - application to Mycobacterium tuberculosis.

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved.