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. 2006 Oct 11:7:34.
doi: 10.1186/1471-2199-7-34.

N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination

Valery N Urakov  1 , Igor A Valouev, Natalia V Kochneva-Pervukhova, Anna N Packeiser, Alexander Yu Vishnevsky, Oleg O Glebov, Vladimir N Smirnov, Michael D Ter-Avanesyan
Affiliations

N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination

Valery N Urakov et al. BMC Mol Biol. .

Abstract

Background: Termination of translation in eukaryotes requires two release factors, eRF1, which recognizes all three nonsense codons and facilitates release of the nascent polypeptide chain, and eRF3 stimulating translation termination in a GTP-depended manner. eRF3 from different organisms possess a highly conservative C region (eRF3C), which is responsible for the function in translation termination, and almost always contain the N-terminal extension, which is inessential and vary both in structure and length. In the yeast Saccharomyces cerevisiae the N-terminal region of eRF3 is responsible for conversion of this protein into the aggregated and functionally inactive prion form.

Results: Here, we examined functional importance of the N-terminal region of a non-prion form of yeast eRF3. The screen for mutations which are lethal in combination with the SUP35-C allele encoding eRF3C revealed the sup45 mutations which alter the N-terminal domain of eRF1 and increase nonsense codon readthrough. However, further analysis showed that synthetic lethality was not caused by the increased levels of nonsense codon readthrough. Dominant mutations in SUP35-C were obtained and characterized, which remove its synthetic lethality with the identified sup45 mutations, thus indicating that synthetic lethality was not due to a disruption of interaction with proteins that bind to this eRF3 region.

Conclusion: These and other data demonstrate that the N-terminal region of eRF3 is involved both in modulation of the efficiency of translation termination and functioning of the eRF1/eRF3 complex outside of translation termination.

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Figures

Figure 1
Figure 1
The sup45-sl23ts mutation is lethal in the strain 33G-D373-rSL23-ΔS35, carrying the SUP35-C or SUP35-MC alleles. The LEU2 plasmids with SUP35-C or SUP35-MC can not replace the resident URA3 plasmid pRS316-SUP35 carrying wild type SUP35 in the strain 33G-D373-rSL23-ΔS35. Transformants were incubated on 5FOA-containing medium for 3 days. Plasmids: SUP35-C, pRS315-SUP35C; SUP35-MC, pRS315-SUP35MC; SUP35, pRS315-SUP35; empty vector, pRS315.
Figure 2
Figure 2
Extra copies of sup45-sl23ts improve growth of the strain 33G-D373-rSL23 carrying the chromosomal sup45-sl23ts mutation at 37°C (A), but do not influence nonsense codon readthrough (B). Empty vector, YEplac181; multi-sup45-sl23, YEplac181-sup45-sl23. Photos were taken after incubation of transformants on leucine omission medium for 4 days. The strain 33G-D373-rSL23 carrying the plasmids indicated above, was transformed with either one of the pUKC815, 817, 818, 819 plasmids and grown in medium selective for plasmids to mid log phase. Then appropriate aliquots of yeast culture were taken and β-galactosidase activity was assayed. Suppression efficiency (average from three independent transformants, each in three parallels) was estimated as described [44].
Figure 3
Figure 3
Cell morphology defects of the sup45 mutants incubated at 37°C. The strain 33G-D373-ΔS45 disrupted for SUP45 and bearing the plasmids pRS315-sup45-sl23 (sup45-sl23), pRS315-sup45-36 (sup45-36) or pRS315-SUP45 (SUP45), was grown in liquid YPD medium for 15 h at 37°C. Bar = 6 μm.
Figure 4
Figure 4
Compensatory mutations in plasmid SUP35-C restore viability of the strain carrying the sup45-36ts and SUP35-C alleles. The sup45-36ts mutant 33G-D373-r36-ΔS35 disrupted for SUP35 and carrying the SUP35 URA3 pRS316-SUP35 plasmid was transformed with either one of the LEU2 plasmids pRS315-SUP35 (SUP35), pRS315-SUP35C (SUP35-C), pRS315-SUP35-C11 (SUP35-C11), pRS315-SUP35-C14 (SUP35-C14), pRS315-SUP35-C42 (SUP35-C42) or pRS315 (empty vector). Transformants were incubated on 5FOA-containing medium for 3 days.
Figure 5
Figure 5
Influence of the compensatory mutations in SUP35-C on the levels of nonsense codon readthrough in the sup45-sl23ts 33G-D373-rSL23 (A) and sup45-36ts 33G-D373-r36 (B) mutants. Plasmids: SUP35-C, pRS315-SUP35C; SUP35-C11, pRS315-SUP35C11; SUP35-C14, pRS315-SUP35C14; SUP35-C42, pRS315-SUP35C42. Suppression efficiency (average from three independent transformants, each in three parallels) was estimated with the use of the pUKC815, 817, 818, 819 plasmids.
Figure 6
Figure 6
Interaction between the sup45ts and SUP35-C alleles does not result in increase of the efficiency of nonsense codon readthrough. Data for the 33G-D373-rSL23 (sup45-sl23ts SUP35) and 33G-D373-rSL23-r35C (sup45-sl23ts SUP35-C) strains (A), as well as for 33G-D373-r36 (sup45-36ts SUP35) and 33G-D373-r36-r35C (sup45-36ts SUP35-C) strains (B) are presented. All strains carrying the pCM183-SUP45 plasmid (tetO2-SUP45) were transformed with either one of the pUKC815, 817, 818, 819 plasmids and grown in tryptophane omission medium to mid log phase. Then the cells were collected, resuspended in analogous medium containing 10 μg/ml doxycycline. After 17 h of incubation in this medium, appropriate aliquots of yeast culture were taken and β-galactosidase activity was assayed. All data represent an average of at least three independent experiments.
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