Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

Biomimetic modeling of the nitrogen-centered radical postulated to occur during the inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N·) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N·-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H 2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.