Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Master of Science in Space & Planetary Sciences (MS)

Degree Level

Graduate

Department

Graduate School

Advisor

Daniel J. Lessner

Committee Member

Ralph L. Henry

Second Committee Member

Timothy A. Kral

Third Committee Member

John C. Dixon

Abstract

The primordial Earth which hosted the first forms of life was an environment free of oxygen. Early organisms utilized metabolisms dependent upon anaerobic conditions and incorporated systems to which oxygen is deleterious. As the content of oxygen in Earth's atmosphere increased, anaerobic organisms had to acquire methods to sense and combat oxygen and reactive oxygen species. Several mechanisms were advantageous to such anaerobic organisms which correlated transcriptional processes with the redox state of the cell so that energy may be conserved and oxygen stress recovery genes activated during periods of oxidative stress. Iron sulfur (Fe-S) cluster cofactors incorporated within RNA polymerase (RNAP) may sense oxygen to globally regulate transcription. Methanosarcina acetivorans, a methanogenic archaeon, offers an opportunity to study an RNAP with two Fe-S clusters within an organism of a phylogenetically and metabolically diverse group. An in vitro transcription system for M. acetivorans could be used to investigate the effects Fe-S cluster integrity on RNAP activity, which would require the components involved in promoter-specific transcription: RNAP, TATA-binding protein (TBP), and transcription factor B (TFB). This work describes the purification of M. acetivorans TBP and TFB for the development of such a system. M. acetivorans also possesses the methanogen-specific redox-sensitive transcriptional regulator MsvR. This work provides evidence of a physiologically-relevant reducing partner for MsvR. As of yet, M. acetivorans MsvR has only been observed to bind to its own gene. This work investigates the other potential gene targets for MsvR.

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