Abstract
In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm(5) ) and 5-methylcarboxymethyl (mcm(5) ) side chains on uridines present at the wobble position (U34 ) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5' AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.
Highlights
The Elongator complex of Saccharomyces cerevisiae was first reported to be associated with the hyper phosphorylated elongating form of RNA polymerase II (Pol II) and three proteins (Elp1p, Elp2p, and Elp3p) constituted the identified complex (Otero et al 1999)
Translation of Elp1p starts at the second AUG of the ELP1 open reading frame (ORF)
When ATG1 was mutated to TTG, the expression level of Elp1p was similar as wild type, whereas when
Summary
The Elongator complex of Saccharomyces cerevisiae was first reported to be associated with the hyper phosphorylated elongating form of RNA polymerase II (Pol II) and three proteins (Elp1p, Elp2p, and Elp3p) constituted the identified complex (Otero et al 1999). Loss of Urm1p or Kti11p increases the amount of truncated Elp1p, abolishes formation of mcm or s2 group of mcm5s2U34, and makes cells resistant to γ-toxin, suggesting that both Kti11p and Urm1p influenced Elp1p proteolysis and are required for proper Elongator function/ regulation (Fichtner et al 2003). It was not established how the truncated form of Elp1p was generated, neither was the exact truncation site determined. We found that appearance of N-terminal truncated Elp1p is a preparation artifact which can be circumvented using an alternative protein extraction method
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
