Archaeal Transcriptional Regulation of Catabolic Carbon Monoxide Dehydrogenase in Methanosarcina Species

Title of Document: ARCHAEAL TRANSCRIPTIONAL REGULATION OF CATABOLIC CARBON MONOXIDE DEHYDROGENASE IN METHANOSARCINA SPECIES Kimberly Lynn Anderson, Doctor of Philosophy, 2009 Directed By: Professor Kevin R. Sowers, Marine Estuarine Environmental Sciences In Archaea, the basal transcription machinery is eukaryotic-like, but some components, such as activator and repressor proteins, are bacteria-like. To further gain knowledge into cellular processes of Archaea, the genome of Methanosarcina thermophila was searched for helicase genes. A homolog of yeast RAD25, a gene with helicase and nucleotide excision repair (NER) abilities, was isolated. M. thermophila rad25 has the domains for helicase activity, but the C-terminal end is truncated, indicating that this protein mostly likely does not function in NER. After overexpression, helicase activity assays of Rad25 indicated that it might have helicase activity; however, there appeared to be contaminating proteins in the purification, so it was not possible to assign the activity only to Rad25. Additional work is necessary to characterize this protein. To investigate transcription, catabolic gene regulation was studied, specifically regulation of carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) from Methanosarcina species. The regions upstream of the transcriptional start site, as well as the 5’ leader region of cdhA, were investigated for trans factors and cis elements that might be involved in regulation. Experiments revealed that regulation of cdhABCDE does not appear to involve trans factors upstream of the transcriptional start site. However, deletion analysis indicated that the 5’ leader region does have a role in regulation. Comparing the protein levels to the mRNA levels revealed there was no significant difference between the two, indicating that translational regulation was not a factor. Other experiments ruled out differential mRNA stability as a factor in regulation. A region located between +358 and +405 was important in transcriptional regulation, indicating that regulation occurred at the level of transcription elongation. A model for regulation of catabolic CODH/ACS by differential elongation is proposed. Although 5’ leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential elongation. Identifying regulatory mechanism(s) of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by the methanoarchaea to efficiently direct carbon and electron flow during biomass conversion to methane. ARCHAEAL TRANSCRIPTIONAL REGULATION OF CATABOLIC CARBON MONOXIDE DEHYDROGENASE IN METHANOSARCINA SPECIES