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

Interakcija podjedinice H arhejske RNA-polimeraze (RpoH) i seril-tRNA-sintetaze metanogenog tipa iz arheje Methanothermobacter thermautotrophicus (MtSerRS) detektirana je ranije u laboratoriju sustavom dvaju hibrida. Asocijacija MtSerRS:RpoH vrlo je zanimljiva budući da se radi o proteinima uključenim u proces koji je od vitalne važnosti za život stanice – ekspresiju gena. Funkcija arhejske RNA-polimeraze je transkripcija svih gena, dok MtSerRS aminoacilira tRNASer serinom te na taj način opskrbljuje ribosom supstratima za elongacijsku fazu translacije. U svrhu potvrde stvaranja kompleksa MtSerRS:RpoH tehnikama in vitro, bilo je potrebno pročistiti pojedine proteine u dovoljnim količinama. Zato je gen rpoH umnožen lančanom reakcijom polimeraze i ugrađen u vektor pod kontrolom jakog T7 promotora. Optimiran je sustav za prekomjernu ekspresiju i pročišćavanje RpoH iz bakterije E.coli, a zatim je RpoH pročišćen u preparativnoj količini. Također, prekomjerno je eksprimirana i pročišćena MtSerRS. Interakcija dvaju proteina istražena je gel-retardacijskom elektroforezom i supročišćavanjem kompleksa na koloni. Preliminarni rezultati dali su naznaku stvaranja kompleksa na nativnom gelu, međutim supročišćavanjem kompleksa na koloni asocijacija MtSerRS:RpoH nije detektirana.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

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

Interakcija podjedinice H arhejske RNA-polimeraze (RpoH) i seril-tRNA-sintetaze metanogenog tipa iz arheje Methanothermobacter thermautotrophicus (MtSerRS) detektirana je ranije u laboratoriju sustavom dvaju hibrida. Asocijacija MtSerRS:RpoH vrlo je zanimljiva budući da se radi o proteinima uključenim u proces koji je od vitalne važnosti za život stanice – ekspresiju gena. Funkcija arhejske RNA-polimeraze je transkripcija svih gena, dok MtSerRS aminoacilira tRNASer serinom te na taj način opskrbljuje ribosom supstratima za elongacijsku fazu translacije. U svrhu potvrde stvaranja kompleksa MtSerRS:RpoH tehnikama in vitro, bilo je potrebno pročistiti pojedine proteine u dovoljnim količinama. Zato je gen rpoH umnožen lančanom reakcijom polimeraze i ugrađen u vektor pod kontrolom jakog T7 promotora. Optimiran je sustav za prekomjernu ekspresiju i pročišćavanje RpoH iz bakterije E.coli, a zatim je RpoH pročišćen u preparativnoj količini. Također, prekomjerno je eksprimirana i pročišćena MtSerRS. Interakcija dvaju proteina istražena je gel-retardacijskom elektroforezom i supročišćavanjem kompleksa na koloni. Preliminarni rezultati dali su naznaku stvaranja kompleksa na nativnom gelu, međutim supročišćavanjem kompleksa na koloni asocijacija MtSerRS:RpoH nije detektirana.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

The aminoacyl-tRNA Synthetase Data Bank (AARSDB).

Aminoacyl-tRNA synthetases (AARSs) are the key components of the protein biosynthesis machinery. They are responsible for maintaining the fidelity of transfer of genetic information from DNA into protein. The database is a compilation of amino acid sequences of all aminoacyl-tRNA synthetases known to date. It contains 422 primary structures of the AARSs available as separate entries or alignments of related proteins. The database is available via the World Wide Web at http://rose.man.poznan.pl/aars/index.htm