Naslov
Quantitative analysis of the Escherichia coli proteome in the absence of LeuRS proofreading

Autori
Cvetešić, Nevena ; Soufi, Boumediene ; Šemanjski, Maja ; Maček, Boris ; Gruić-Sovulj, Ita

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
25th tRNA Conference 2014 Abstract Book / Drainas, Denis ; Stathopoulos Constantinos - , 2014, 161-161

Skup
25th tRNA Conference 2014

Mjesto i datum
Kyllíni, Grčka, 21.09.2014. - 25.09.2014

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
leucyl-tRNA synthetase; norvaline; mass spectrometry; SILAC

Sažetak
Leucyl-tRNA synthetase (LeuRS) efficiently activates norvaline, a non-canonical amino acid that accumulates in Escherichia coli during micro- aerobic growth. Remarkably, in spite of the prevailing opinion that LeuRS does not discriminate well against the non-cognate isoleucine, we have recently demonstrated that isoleucine does not pose a threat for LeuRS fidelity in vitro or in vivo. This points to norvaline as a prime candidate for proofreading activity. To establish an impact of translational quality control on maintaining a functional proteome and also to quantitate the level of mistranslation triggered by inactivation of LeuRS editing, the proteomes of the LeuRS deacylation- defective or wild-type E. coli strain grown under aerobic or micro-aerobic conditions were quantitated and compared using stable isotope labeling by amino acids in cell culture (SILAC). By this approach, we unambiguously demonstrate that LeuRS editing is indeed essential under micro-aerobic conditions to prevent non-canonical mistranslation. E. coli strain that lacks LeuRS post-transfer editing substitutes leucine with norvaline throughout the proteome, resulting in approximately 14 % mistranslation level. In contrast, no significant isoleucine misincorporation is detected in the same strain even when non-physiologically high concentration of isoleucine is added to the growth media. Colony-forming unit assay reveals that norvaline misincorporation is followed by a drop in cell viability, confirming the importance of LeuRS editing. To the best of our knowledge, this study provides the first comprehensive quantitative analysis of mistranslation using SILAC methodology and high resolution mass spectrometry. It also characterizes the completely unknown scale and dynamics of the norvalylated proteome.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA