Archaeal RNA polymerase subunit H: from cloning to investigation of interaction with atypical seryl-tRNA synthetase

Marasović, Mirela (2011) Archaeal RNA polymerase subunit H: from cloning to investigation of interaction with atypical seryl-tRNA synthetase. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

A yeast two hybrid screen performed earlier in our laboratory revealed interaction of Archaeal RNA polymerase subunit H (RpoH) and methanogenic-type seryl-tRNA synthetase from Archaea Methanothermobacter thermautotrophicus (MtSerRS). Association MtSerRS:RpoH is very interesting since both proteins are involved in a process vital for cell's life – expression of genes. Archaeal RNA polymerase transcribes genes while MtSerRS aminoacylates tRNASer with serine and supplies ribosome with substrates for elongation phase of translation. In order to confirm the formation of MtSerRS:RpoH complex by in vitro methods, it was nessesary to obtain the individual proteins in sufficient amounts. Therefore, rpoH gene was amplified using PCR and cloned in a vector under control of T7 strong promoter. The system for overexpression and purification of RpoH in Escherichia coli was oprimized, and then RpoH was purified for preparative purposes. Also, MtSerRS was overexpressed and purified. The interaction between two proteins was investigated by gel-retardation electrophoresis and pull-down assay. Preliminary results indicate complex formation on native gel, but the association MtSerRS:RpoH was not detected by pull-down assay.

Item Type: Thesis (Diploma thesis)
Keywords: Archaea, Methanothermobacter thermautotrophicus, methanogenic-type seryl-tRNA synthetase, RNA polymerase subunit H, gel-retardation electrophoresis, pull-down assay
Supervisor: Weygand-Đurašević, Ivana
Date: 2011
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 18 Jul 2014 11:31
Last Modified: 18 Jul 2014 11:31
URI: http://digre.pmf.unizg.hr/id/eprint/2524

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