| dc.description.abstract | There are two overlapping but divergently oriented promoters, momP1 and momP2. E. coli RNA polymerase is unable to bind to momP1 on its own. A possibility of DNA curvature in the promoter region preventing RNAP occupancy is addressed in Chapter 2. This chapter describes the experiments designed to delineate the role of the T6 run within the spacer region of the momP1 promoter in mom gene transcription. To investigate the influence of the T6-run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline [(OP^Cu)] and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared to the wild type, the mutants showed an increase in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts as a negative element in mom operon transcription initiation.
Regulation of the mom gene at the transcriptional level is brought about by the phage-encoded transactivator protein C. In Chapter 3, the mechanism by which C protein acts as a transcriptional activator at momP1 has been addressed. The momP1 promoter has poor -10 and -35 elements separated by a 19 bp suboptimal spacer region. These features make this promoter dependent on an activator for its optimum activity. In vivo DMS footprinting studies reveal C protein-mediated distortion of its specific site at the mom regulatory region. Using a coupled topoisomerase assay, C protein-induced unwinding of DNA is demonstrated. This C-mediated untwisting seems to be localized to the 3’ flanking region of the C binding site, adjacent to and overlapping the -35 element of momP1. These results suggest that C protein-mediated torsional changes could be reorienting the -10 and -35 elements to a favorable conformation for RNA polymerase occupancy at the mom promoter. A working model to explain the C-mediated transactivation process is proposed at the end of the chapter.
Chapter 4 describes the experiments to test the C-mediated unwinding model. Various spacer mutants of momP1 were constructed and promoter strength was analyzed both in the absence and presence of C protein. There was no appreciable change in the low-level constitutive activity of all the promoters having spacer length mutations. As described in Chapter 2, the presence of the negative element in the form of the T6 run within the spacer region of the momP1 promoter explains this result. Disruption of the negative element led to an increase in the constitutive promoter activity of the 17 bp spacer mutant. The response to C-mediated transactivation was dependent on the spacer length of the mutant promoters. The mutant with 17 bp spacing between the promoter elements showed less activity in the presence of C protein as compared to its basal level, which supports the unwinding model. These data, along with the experiments using synthetic promoters designed to test the unwinding model, support a role for C-mediated unwinding in mom transcription activation.
Apart from the transactivator protein C, two other host-encoded proteins, OxyR and Dam methylase, also play important roles in the regulation of the mom promoter at the transcriptional level. An in vivo analysis has been described in Chapter 5 to understand the interplay of these factors in the regulation of both momP1 and momP2 promoters. Promoter strength was assessed by estimating the amount of momP1- and momP2-specific transcripts under different conditions. The significance of the role of these regulators has been discussed in the context of bacteriophage Mu biology.
The Appendix section describes the development of a novel in vivo footprinting technique using 1,10-phenanthroline complexed with copper. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is used: (i) to detect structural alteration in DNA as a result of mutation, (ii) mapping of contacts of site-specific DNA-binding protein, (iii) promoter occupancy by RNA polymerase, and (iv) molecular interactions during transcription initiation. The results indicate that in vivo [(OP^Cu)] probing could be a very useful tool in the post-genomic era for studying a variety of important cellular processes involving DNA-protein interactions. | |
