An early step in wobble uridine tRNA modification requires the Elongator complex

Abstract

Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1-ELP6) genes or the three killer toxin insensitivity (KTI11-KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1-ELP6 and KTI11-KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1-elp6 and kti11-kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1-ELP6 or KTI11-KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

FIGURE 1.

Structure of 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), 5-carbamoyl-methyluridine (ncm⁵U), and 5-carboxymethyluridine (cm⁵U). R represents ribose.

FIGURE 2.

Strains with elp and kti mutations confer a loss-of-suppression phenotype. Approximately 10⁴ cells of wild type (W303-1B), SUP4 (UMY2893), SUP4 elp1-elp6 (UMY2916, UMY3039, UMY3040, UMY2912, UMY2914, UMY2918, UMY2920, UMY2922), and SUP4 kti11-kti13 (UMY2936, UMY2938, UMY2940) strains were spotted on SC, SC-Ade, or SC-Arg+Can plates and incubated for 3 d at 30°C.

FIGURE 3.

An elp3-null mutant is lacking mcm⁵ and ncm⁵ side chains at wobble uridines. HPLC analysis of modified tRNA nucleosides from SUP4 (UMY2893, left panels) and SUP4 elp3::KanMX4 (UMY2916, right panels) strains. (A) Analysis of modified nucleosides in tRNA^Glu_mcm⁵s²UUC. The part of the chromatogram between retention times 33 and 54 min is shown. Arrows indicate the expected retention time of s²U (left panel) and mcm⁵s²U (right panel). (B) Analysis of modified nucleosides in tRNA^Arg_mcm⁵UCU. The part of the chromatogram between retention times 33 and 45.5 min is shown. The arrow indicates the expected retention time of mcm⁵U (right panel). (C) Analysis of modified nucleosides in tRNA^Pro _ncm⁵UGG. The part of the chromatogram between retention times 10 and 20 min is shown. The arrow indicates the expected retention time of ncm⁵U (right panel). The small peak at this position represents an unrelated compound with a spectrum different from that of ncm⁵U.

FIGURE 4.

Elp1p and Elp3p coimmunoprecipitate tRNA^Glu_UUC. (A) T7-transcribed ³²P-labeled tRNA^Glu _UUC was mixed with extracts from wild-type, ELP1-(HA)₃, ELP3-(HA)₃, or ELP5-(HA)₃ strains. After UV-cross-linking, immunoprecipitation using agarose conjugated anti-HA antibodies was performed. The immunoprecipitated material was digested with proteinase K and analyzed on a denaturing polyacrylamide gel. The signals were quantified and normalized to the background signal in the control, which was set to 1. (B) T7-transcribed ³²P-labeled tRNA^Glu_UUC or tRNA^Met_i was mixed with extracts from TIF4632-(HA)₃, ELP1-(HA)₃, ELP3-(HA)₃, or ELP5-(HA)₃ strains. The experiment and quantifications were performed as described in A.