Research of translational autoregulation of the sgm gene in vitro

[T. Ilić-Tomić]

136, [10] listova s tablama

Molekularna biologija / Molecular biology

Proizvođač aminoglikozidnog antibiotika G-52, Micromonospora zionensis poseduje sgm (sisomicin-gentamicin metilaza) gen koji kodira 16S rRNK metiltransferazu čijom se aktivnošću ostvaruje rezistencija na 4,6 disupstituisane aminoglikozidne antibiotike. Na osnovu rezultata dobijenih in vivo korišćenjem sgm-lacZ genskih i operonskih fuzija, u heterologom domaćinu E. coli, postavljena je hipoteza o translacionoj autoregulaciji sgm gena. Hipoteza se z;asniva na činjenici da je potreno malo molekula Sgm metiltransferaze u ćeliji za potpunu modifikaciju ribozoma i da se ovaj enzim vezuje za sopstvenu iRNK sprečavajući dalju inicijaciju translacije kada su svi ribozomi metilovani. In vitro analiza traslacione autoregulacije sgm gena je urađena u cilju razrešenja mehanizma interakcije Sgm protein-sopstvena iRNK. U tu svrhu, Sgm protein je eksprimiran i prečišćen metal afinitetnom hromatografljom. Metilacionim esejom je indirektno pokazano da je mesto delovanja Sgm metiltransferaze G1405 u 16S rRNK. Rekombinantni Sgm protein je korišćen u vezivanju za regulatome regione u 5’ UTR sgm- iRNK molekula u EMSA eksperimentima. Takođe je korišćen u ,,toeprint“ eseju da bi se determinisao mehanizam translacione represije. Ovi rezultati su pokazali da Sgm protein ne poseduje visok afmitet i specifičnost za sopstvenu iRNK, što je navelo na zaključak da je mehanizam dejstva mnogo složeniji od onog iznetog u hipotezi. Takođe, učinjen je pokušaj da se translaciona represija dokaže u „cell free“ sistemu E. coli za in vitro transkripciju i translaciju. Pokazano je da se sgm eksprimira u ovakvom sistemu samo ukoliko je pod kontrolom T5 promotora i signala za inicijaciju translacije (RBS i ATG) iz samog ekspresionog vektora pQE 30.

In Micromonospora zionensis, a producer of G-52 antibiotic, the sgm gene encodes 16S rRNA methyltransferase and thus provide resistance against 4,6- disubstituted aminoglycosides antibiotics. The hypothesis of autoregulation of the sgm gene expression at the translational level, was proposed accoding to in vivo results, obtained by using sgm-lacZ gene and operon fusions, in heterologous host such as E. coli. The hypothesis is based on the fact that a relatively few molecules of enzymes are sufficient for complete modification of the target (i.e., 16S rRNA). When all ribosomes are protected, unnecessary translation is prevented by binding to its own mRNA. In order to resolve the mechanism of the Sgm protein-mRNA interaction, in vitro analysis of translational autoregulation of the sgm gene was performed. For that purpose, recombinant Sgm protein was expressed and purified by metal-affmity chromatography. It was shown in in vitro methylation assay, that Sgm methyltransferase acts at the residue G1405 within 16S rRNA. Recombinant Sgm protein was used in binding reactions with regulatory regions within its own mRNA, in mobility shift assays. Also, purified Sgm was used in toeprint assays in order to determine the mechanism of translational repression. The obtained results demonstrated that Sgm does not_have high affinity for its own mRNA as well as that the mechanism of regulation of the sgm gene is more complex then in previously proposed hypothesis. Furthermore, the E. coli „cell free system for coupled in vitro transcription and translation was_used_in analysis of regulation of the sgm gene. It was shown that .vgm expression is possible only under control of T5 promoter and signals for initiation of translation from the commercial vector pQE 30.

translaciona autoregulacija, Sgm metiltransferaza, rezistencija, 16S rRNK

translational autoregulation, Sgm methyltransferase, resistance, 16S rRNA

577.217(043.3)

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