Plasmids used in this study
| Name | Genotype | Reference/source |
| pADH181 pA-TEV-ctrsa4 Δ1–29 | 2µ, LEU2, AmpR, PADH1 pA-TEV-ctrsa4 30–517aa, Chaetomium thermophilum Rsa4 | This study |
| pASΔΔ NSA2 | 2µ, TRP1, PADH1 Gal4-DNA BD-NSA2 | This study |
| pASΔΔ nsa2 C (ΔN4) | See above, pASΔΔ Nsa2 141–261 aa | This study |
| pASΔΔ nsa2 N (ΔC4) | See above, pASΔΔ Nsa2 1–144 aa | This study |
| pASΔΔ nsa2 ΔN1 | See above, pASΔΔ Nsa2 77–261 aa | This study |
| pASΔΔ nsa2 ΔN2 | See above, pASΔΔ Nsa2 35–261 aa | This study |
| pEcOmeTyr/ectRNACUA | CEN, TRP1, expression of amber suppressor ectRNACUA and aminoacyl ectRNA-synthetase | Chin et al., 2003 |
| pET Duet1 rsa4 Δ136 | AmpR, PT7 rsa4 ΔN 136 aa | This study |
| pET24d HIS6-TEV-ctnsa2 1–84 | KanR, PT7 HIS6-TEV-ctnsa2 1–84 aa Chaetomium thermophilum Nsa2 | This study |
| pET24d HIS6-TEV-ctnsa2 168–261 | KanR, PT7 HIS6-TEV-ctnsa2 168–261 aa Chaetomium thermophilum Nsa2 | This study |
| pET24d HIS6-TEV-RSA4 | KanR, PT7 HIS6-TEV-RSA4 | This study |
| pET24d HIS6-TEV-RSA4 | KanR, PT7 HIS6-TEV-RSA4 | This study |
| pET24d HIS6-TEV-rsa4 b1* | KanR, PT7 HIS6-TEV-rsa4 T175R,T177R | This study |
| pET24d HIS6-TEV-rsa4 b8* | KanR, PT7 HIS6-TEV-rsa4 K130E,R134E | This study |
| pET24d HIS6-TEV-rsa4 Y448E | KanR, PT7 HIS6-TEV-rsa4 Y448E | This study |
| pETM43 MBP | KanR, empty vector, PT7, MBP control | EMBL Core facility |
| pETM43 MBP-PRE-NSA2 | KanR, PT7 MBP-PRE-NSA2 | This study |
| pETMBPxray_V43 | KanR, PT7, MBP (optimized for carrier-driven crystallization: D82A, K83A, K239A, E359A, K362A, D363A)-linker (AAAA) NcoI | This study |
| pETMBPxray_V43 MBP-nsa2 81–101 | See above, Nsa2 peptide: 81–101 aa | This study |
| pETMBPxray_V43 MBP-nsa2 84–96 | See above, Nsa2 peptide: 84–96 aa | This study |
| pG4ADHAN RSA4 | CEN, LEU2, PADH1 GAL4-AD-RSA4 1–515 aa | This study |
| pGADT7 rsa4 MIDO | 2µ, LEU2, PADH1 GAL4-AD-rsa4 1–154 aa | Ulbrich et al., 2009 |
| pHAC111 NOG1 | LEU2, NOG1-HA3 | Honma et al., 2006 |
| pHAC111 nog1-11 | LEU2, nog1-11-HA3 | Honma et al., 2006 |
| pnatNT2 PRSA4 NTAP-FLAG | CEN, AmpR, PTEF1 natNT2 TADH1, PRSA4 2× pA-TEV-CBP-FLAG, integration cassette | This study |
| pRS314 NSA2 | CEN, TRP1, AmpR, PNSA2 NSA2 | This study |
| pRS314 nsa2-1 | CEN, TRP1, AmpR, PNSA2 nsa2-1, point mutation in STOP leads to elongation of Nsa2p by 17 aa: RAKKCSVFFFFYLDGSN* | This study |
| pRS314 nsa2-3 | CEN, TRP1, AmpR, PNSA2 nsa2 N203K | This study |
| pRS314 nsa2-9 | CEN, TRP1, AmpR, PNSA2 nsa2 S177R,H186P | This study |
| pRS314 RSA4 | CEN, TRP1, AmpR, PRSA4 RSA4 | Ulbrich et al., 2009 |
| pRS314 rsa4-1 | CEN, TRP1, AmpR, PRSA4 rsa4-1 | Ulbrich et al., 2009 |
| pRS315 IPI3 | CEN, LEU2, AmpR, PIPI3 IPI3 | This study |
| pRS315 ipi3-2 | CEN, LEU2, AmpR, PIPI3 ipi3-2 | This study |
| pRS315 NSA2 | CEN, LEU2, AmpR, PNSA2 NSA2 | This study |
| pRS315 nsa2 Y90A | CEN, LEU2, AmpR, PNSA2 nsa2 Y90A | This study |
| pRS315 nsa2 Y90F | CEN, LEU2, AmpR, PNSA2 nsa2 Y90F | This study |
| pRS315 rix1-1 | CEN, LEU2, AmpR, PRIX1 rix1-1 | Baßler et al., 2001 |
| pRS315 RSA4 | CEN, LEU2, AmpR, PRSA4 RSA4 | Ulbrich et al., 2009 |
| pRS315 rsa4-1 | CEN, LEU2, AmpR, PRSA4 rsa4-1 | Ulbrich et al., 2009 |
| pRSF Duet1 GST-TEV-NSA2 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-NSA2 + PT7 HIS6-TEV-RSA4 | This study |
| pRSF Duet1 GST-TEV-NSA2 Δ85–98 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-nsa2 Δ 85–98 aa + PT7 HIS6-TEV-RSA4 | This study |
| pRSF Duet1 GST-TEV-NSA2 Δ86–90 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-nsa2 Δ 86–90 aa + PT7 HIS6-TEV-RSA4 | This study |
| pT7 HIS6-rsa4 ΔN | KanR, PT7 HIS6-RSA4 ΔN 112–516 aa | This study |
| pT7 HIS6-TEV-rsa4 ΔN26 | KanR, PT7 HIS6-TEV-rsa4 27–515 aa | This study |
| pTD1-1 | 2µ, LEU2, GAL4 AD-SV40 large T-antigen (84-708 aa), pACT2 | Takara Bio Inc. |
| pUN100 RIX1 | CEN, LEU2, AmpR, PRIX1 RIX1 | Baßler et al., 2001 |
| pVA3-1 | 2µ, TRP1, GAL4 DNA BD-murine p53 (72–390 aa), pGBT9 | Takara Bio Inc. |
| YCplac111 NSA2-FTpA | CEN, LEU2, AmpR, PNSA2 NSA2-LINKER-FLAG-TEV-pA, Linker:ASSYTAPQPGLGGS | This study |
| YCplac111 nsa2-FTpA Δ85–98 | See above, PNSA2 nsa2-L-FTpA, Δ 85–98 aa | This study |
| YCplac111 nsa2-FTpA Δ86–90 | See above, PNSA2 nsa2-L-FTpA, Δ 86–90 aa | This study |
| YCplac111 PGAL NSA2-FTpA | CEN, LEU2, AmpR, PGAL1-10 NSA2-LINKER-FLAG-TEV–pA,Linker:ASSYTAPQPGLGGS | This study |
| YCplac111 PGAL nsa2-FTpA Δ85–98 | See above, PGAL1-10 nsa2-L-FTpA, Δ 85–98 aa | This study |
| YCplac111 PGAL nsa2-FTpA Δ86–90 | See above, PGAL1-10 nsa2-L-FTpA, Δ 86–90 aa | This study |
| YCplac111 RPL5 | CEN, LEU2, AmpR, PRpl5 RPL5 | This study |
| YCplac111 rpl5 Δloop2 | See above, Δ 122–138 aa | This study |
| YCplac111 rpl5 Δloop2+3 | See above, Δ 122–138 aa, Δ185–198>R aa | This study |
| YCplac111 rpl5 Δloop3 | See above, Δ 185–198>R aa | This study |
| YCplac111 RPL5-FTpA | CEN,LEU2,AmpR,PRPL5 RPL5-5xGA-FTpA | This study |
| YCplac111 rpl5-FTpA Δloop2 | See above, Δ 122–138 aa | This study |
| YCplac111 rpl5-FTpA Δloop2+3 | See above, Δ 122–138 aa, Δ 185–198>R aa | This study |
| YCplac111 rpl5-FTpA Δloop3 | See above, Δ 185–198>R aa | This study |
| YCplac111 RPL5-GFP | CEN, LEU2, AmpR, PRPL5 RPL5-GFP | This study |
| YCplac111 rpl5-GFP Δloop2+3 | See above, Δ 122–138 aa, Δ 185–198>R aa | This study |
| YCplac111 TAP-FLAG-rsa4 b1* | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 K130E,R134E | This study |
| YCplac111 TAP-FLAG-rsa4 b8* | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 T175R,T177R | This study |
| YCplac111 TAP-FLAG-rsa4 Y448E | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 Y448E | This study |
| YCplac22 NSA2-FTpA | CEN, TRP1, AmpR, PNSA2 NSA2-LINKER-FLAG-TEV-pA, Linker:ASSYTAPQPGLGGS | This study |
| YCplac22 nsa2-FTpA Y90A | See above, PNSA2 nsa2-LINKER-FLAG-TEV-pA, nsa2 Y90A, Linker:ASSYTAPQPGLGGS | This study |
| YEplac112 PGAL1-10 NSA2 | 2µ, TRP1, AmpR, PGAL1-10 NSA2 | This study |
| YEplac112 PGAL1-10 nsa2 Y90A | See above, PGAL1-10 nsa2 Y90A | This study |
| YEplac112 PGAL1-10 RSA4 | See above, PGAL1-10 RSA4 | This study |
| YEplac112 PGAL1-10 rsa4 b1* | See above, PGAL1-10 rsa4 K130E,R134E | This study |
| YEplac112 PGAL1-10 rsa4 b8* | See above, PGAL1-10 rsa4 T175R,T177R | This study |
| YEplac112 PGAL1-10 rsa4 Y448E | See above, PGAL1-10 rsa4 Y448E | This study |
| YEplac181 PGAL1-10 NSA2 PGAL1-10-tc-apt-2×HA-TAG-RPL25-FTpA | 2µ, LEU2, AmpR, PGAL1-10 NSA2 TADH1, PGAL1-10 tetracycline-aptamer-2×HA-amber-RPL25-FLAG-TEV-pA TADH1 | This study |
| YEplac181 PGAL1-10 nsa2 Y90A PGAL1-10-tc-apt-2×HA-TAG-RPL25-FTpA | 2µ, LEU2, AmpR, PGAL1-10 nsa2 Y90A TADH1 PGAL1-10 tetracycline-aptamer-2×HA-amber-RPL25-FLAG-TEV-pA TADH1 | This study |
| Name | Genotype | Reference/source |
| pADH181 pA-TEV-ctrsa4 Δ1–29 | 2µ, LEU2, AmpR, PADH1 pA-TEV-ctrsa4 30–517aa, Chaetomium thermophilum Rsa4 | This study |
| pASΔΔ NSA2 | 2µ, TRP1, PADH1 Gal4-DNA BD-NSA2 | This study |
| pASΔΔ nsa2 C (ΔN4) | See above, pASΔΔ Nsa2 141–261 aa | This study |
| pASΔΔ nsa2 N (ΔC4) | See above, pASΔΔ Nsa2 1–144 aa | This study |
| pASΔΔ nsa2 ΔN1 | See above, pASΔΔ Nsa2 77–261 aa | This study |
| pASΔΔ nsa2 ΔN2 | See above, pASΔΔ Nsa2 35–261 aa | This study |
| pEcOmeTyr/ectRNACUA | CEN, TRP1, expression of amber suppressor ectRNACUA and aminoacyl ectRNA-synthetase | Chin et al., 2003 |
| pET Duet1 rsa4 Δ136 | AmpR, PT7 rsa4 ΔN 136 aa | This study |
| pET24d HIS6-TEV-ctnsa2 1–84 | KanR, PT7 HIS6-TEV-ctnsa2 1–84 aa Chaetomium thermophilum Nsa2 | This study |
| pET24d HIS6-TEV-ctnsa2 168–261 | KanR, PT7 HIS6-TEV-ctnsa2 168–261 aa Chaetomium thermophilum Nsa2 | This study |
| pET24d HIS6-TEV-RSA4 | KanR, PT7 HIS6-TEV-RSA4 | This study |
| pET24d HIS6-TEV-RSA4 | KanR, PT7 HIS6-TEV-RSA4 | This study |
| pET24d HIS6-TEV-rsa4 b1* | KanR, PT7 HIS6-TEV-rsa4 T175R,T177R | This study |
| pET24d HIS6-TEV-rsa4 b8* | KanR, PT7 HIS6-TEV-rsa4 K130E,R134E | This study |
| pET24d HIS6-TEV-rsa4 Y448E | KanR, PT7 HIS6-TEV-rsa4 Y448E | This study |
| pETM43 MBP | KanR, empty vector, PT7, MBP control | EMBL Core facility |
| pETM43 MBP-PRE-NSA2 | KanR, PT7 MBP-PRE-NSA2 | This study |
| pETMBPxray_V43 | KanR, PT7, MBP (optimized for carrier-driven crystallization: D82A, K83A, K239A, E359A, K362A, D363A)-linker (AAAA) NcoI | This study |
| pETMBPxray_V43 MBP-nsa2 81–101 | See above, Nsa2 peptide: 81–101 aa | This study |
| pETMBPxray_V43 MBP-nsa2 84–96 | See above, Nsa2 peptide: 84–96 aa | This study |
| pG4ADHAN RSA4 | CEN, LEU2, PADH1 GAL4-AD-RSA4 1–515 aa | This study |
| pGADT7 rsa4 MIDO | 2µ, LEU2, PADH1 GAL4-AD-rsa4 1–154 aa | Ulbrich et al., 2009 |
| pHAC111 NOG1 | LEU2, NOG1-HA3 | Honma et al., 2006 |
| pHAC111 nog1-11 | LEU2, nog1-11-HA3 | Honma et al., 2006 |
| pnatNT2 PRSA4 NTAP-FLAG | CEN, AmpR, PTEF1 natNT2 TADH1, PRSA4 2× pA-TEV-CBP-FLAG, integration cassette | This study |
| pRS314 NSA2 | CEN, TRP1, AmpR, PNSA2 NSA2 | This study |
| pRS314 nsa2-1 | CEN, TRP1, AmpR, PNSA2 nsa2-1, point mutation in STOP leads to elongation of Nsa2p by 17 aa: RAKKCSVFFFFYLDGSN* | This study |
| pRS314 nsa2-3 | CEN, TRP1, AmpR, PNSA2 nsa2 N203K | This study |
| pRS314 nsa2-9 | CEN, TRP1, AmpR, PNSA2 nsa2 S177R,H186P | This study |
| pRS314 RSA4 | CEN, TRP1, AmpR, PRSA4 RSA4 | Ulbrich et al., 2009 |
| pRS314 rsa4-1 | CEN, TRP1, AmpR, PRSA4 rsa4-1 | Ulbrich et al., 2009 |
| pRS315 IPI3 | CEN, LEU2, AmpR, PIPI3 IPI3 | This study |
| pRS315 ipi3-2 | CEN, LEU2, AmpR, PIPI3 ipi3-2 | This study |
| pRS315 NSA2 | CEN, LEU2, AmpR, PNSA2 NSA2 | This study |
| pRS315 nsa2 Y90A | CEN, LEU2, AmpR, PNSA2 nsa2 Y90A | This study |
| pRS315 nsa2 Y90F | CEN, LEU2, AmpR, PNSA2 nsa2 Y90F | This study |
| pRS315 rix1-1 | CEN, LEU2, AmpR, PRIX1 rix1-1 | Baßler et al., 2001 |
| pRS315 RSA4 | CEN, LEU2, AmpR, PRSA4 RSA4 | Ulbrich et al., 2009 |
| pRS315 rsa4-1 | CEN, LEU2, AmpR, PRSA4 rsa4-1 | Ulbrich et al., 2009 |
| pRSF Duet1 GST-TEV-NSA2 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-NSA2 + PT7 HIS6-TEV-RSA4 | This study |
| pRSF Duet1 GST-TEV-NSA2 Δ85–98 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-nsa2 Δ 85–98 aa + PT7 HIS6-TEV-RSA4 | This study |
| pRSF Duet1 GST-TEV-NSA2 Δ86–90 + HIS6-TEV-RSA4 | KanR, PT7 GST-TEV-nsa2 Δ 86–90 aa + PT7 HIS6-TEV-RSA4 | This study |
| pT7 HIS6-rsa4 ΔN | KanR, PT7 HIS6-RSA4 ΔN 112–516 aa | This study |
| pT7 HIS6-TEV-rsa4 ΔN26 | KanR, PT7 HIS6-TEV-rsa4 27–515 aa | This study |
| pTD1-1 | 2µ, LEU2, GAL4 AD-SV40 large T-antigen (84-708 aa), pACT2 | Takara Bio Inc. |
| pUN100 RIX1 | CEN, LEU2, AmpR, PRIX1 RIX1 | Baßler et al., 2001 |
| pVA3-1 | 2µ, TRP1, GAL4 DNA BD-murine p53 (72–390 aa), pGBT9 | Takara Bio Inc. |
| YCplac111 NSA2-FTpA | CEN, LEU2, AmpR, PNSA2 NSA2-LINKER-FLAG-TEV-pA, Linker:ASSYTAPQPGLGGS | This study |
| YCplac111 nsa2-FTpA Δ85–98 | See above, PNSA2 nsa2-L-FTpA, Δ 85–98 aa | This study |
| YCplac111 nsa2-FTpA Δ86–90 | See above, PNSA2 nsa2-L-FTpA, Δ 86–90 aa | This study |
| YCplac111 PGAL NSA2-FTpA | CEN, LEU2, AmpR, PGAL1-10 NSA2-LINKER-FLAG-TEV–pA,Linker:ASSYTAPQPGLGGS | This study |
| YCplac111 PGAL nsa2-FTpA Δ85–98 | See above, PGAL1-10 nsa2-L-FTpA, Δ 85–98 aa | This study |
| YCplac111 PGAL nsa2-FTpA Δ86–90 | See above, PGAL1-10 nsa2-L-FTpA, Δ 86–90 aa | This study |
| YCplac111 RPL5 | CEN, LEU2, AmpR, PRpl5 RPL5 | This study |
| YCplac111 rpl5 Δloop2 | See above, Δ 122–138 aa | This study |
| YCplac111 rpl5 Δloop2+3 | See above, Δ 122–138 aa, Δ185–198>R aa | This study |
| YCplac111 rpl5 Δloop3 | See above, Δ 185–198>R aa | This study |
| YCplac111 RPL5-FTpA | CEN,LEU2,AmpR,PRPL5 RPL5-5xGA-FTpA | This study |
| YCplac111 rpl5-FTpA Δloop2 | See above, Δ 122–138 aa | This study |
| YCplac111 rpl5-FTpA Δloop2+3 | See above, Δ 122–138 aa, Δ 185–198>R aa | This study |
| YCplac111 rpl5-FTpA Δloop3 | See above, Δ 185–198>R aa | This study |
| YCplac111 RPL5-GFP | CEN, LEU2, AmpR, PRPL5 RPL5-GFP | This study |
| YCplac111 rpl5-GFP Δloop2+3 | See above, Δ 122–138 aa, Δ 185–198>R aa | This study |
| YCplac111 TAP-FLAG-rsa4 b1* | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 K130E,R134E | This study |
| YCplac111 TAP-FLAG-rsa4 b8* | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 T175R,T177R | This study |
| YCplac111 TAP-FLAG-rsa4 Y448E | CEN, LEU2, AmpR, PRSA4 pA-CBP-2×FLAG-rsa4 Y448E | This study |
| YCplac22 NSA2-FTpA | CEN, TRP1, AmpR, PNSA2 NSA2-LINKER-FLAG-TEV-pA, Linker:ASSYTAPQPGLGGS | This study |
| YCplac22 nsa2-FTpA Y90A | See above, PNSA2 nsa2-LINKER-FLAG-TEV-pA, nsa2 Y90A, Linker:ASSYTAPQPGLGGS | This study |
| YEplac112 PGAL1-10 NSA2 | 2µ, TRP1, AmpR, PGAL1-10 NSA2 | This study |
| YEplac112 PGAL1-10 nsa2 Y90A | See above, PGAL1-10 nsa2 Y90A | This study |
| YEplac112 PGAL1-10 RSA4 | See above, PGAL1-10 RSA4 | This study |
| YEplac112 PGAL1-10 rsa4 b1* | See above, PGAL1-10 rsa4 K130E,R134E | This study |
| YEplac112 PGAL1-10 rsa4 b8* | See above, PGAL1-10 rsa4 T175R,T177R | This study |
| YEplac112 PGAL1-10 rsa4 Y448E | See above, PGAL1-10 rsa4 Y448E | This study |
| YEplac181 PGAL1-10 NSA2 PGAL1-10-tc-apt-2×HA-TAG-RPL25-FTpA | 2µ, LEU2, AmpR, PGAL1-10 NSA2 TADH1, PGAL1-10 tetracycline-aptamer-2×HA-amber-RPL25-FLAG-TEV-pA TADH1 | This study |
| YEplac181 PGAL1-10 nsa2 Y90A PGAL1-10-tc-apt-2×HA-TAG-RPL25-FTpA | 2µ, LEU2, AmpR, PGAL1-10 nsa2 Y90A TADH1 PGAL1-10 tetracycline-aptamer-2×HA-amber-RPL25-FLAG-TEV-pA TADH1 | This study |