Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay

Abstract

No-go decay (NGD) is one of several messenger RNA (mRNA) surveillance systems dedicated to the removal of defective mRNAs from the available pool. Two interacting factors, Dom34 and Hbs1, are genetically implicated in NGD in yeast. Using a reconstituted yeast translation system, we show that Dom34:Hbs1 interacts with the ribosome to promote subunit dissociation and peptidyl-tRNA drop-off. Our data further indicate that these recycling activities are shared by the homologous translation termination factor complex eRF1:eRF3, suggesting a common ancestral function. Because Dom34:Hbs1 activity exhibits no dependence on either peptide length or A-site codon identity, we propose that this quality-control system functions broadly to recycle ribosomes throughout the translation cycle whenever stalls occur.

Figure 1

(A) Ribosome stimulation of GTP hydrolysis by Dom34:Hbs1 as visualized by thin layer chromatogram. (B) Native gel analysis of ribosomal termination complexes incubated with indicated components. Stylized diagrams of differentially labeled (³⁵S-Met (blue) or ³²P[CCA]-tRNA (red)) ribosome complexes are on the left. Pth, peptidyl-tRNA hydrolase.

Figure 2

Effects of codon identity and peptide length on Dom34:Hbs1-mediated peptidyl-tRNA release activity. (A) Kinetics of release reactions mediated by Dom34:Hbs1 and eRF1:eRF3 on UAA and CAA A-site-programmed ribosome complexes. k_cat values are reported as mean +/− standard deviation (SD) (N=3). (B) Inhibition of Dom34:Hbs1-mediated reaction by a release-defective eRF1 variant (eRF1[AGQ]) on UAA-programmed ribosomes. Symbols: ■, Dom34/Hbs1 only; ▼, +5uM eRF1 (AGQ); ◆, +20uM eRF1(AGQ); ●, 20uM eRF1(AGQ) only. (C) Rate constants for Dom34:Hbs1-mediated peptidyl-tRNA release on programmed complexes containing Met-tRNA^iMet, di-, tri- or tetra-peptidyl tRNA in the P site (N=4, +/− SD).

Figure 3

Dom34:Hbs1 promotes ribosome subunit dissociation. (A) Native gel analysis demonstrating GTP-dependence of Dom34:Hbs1-mediated products on differentially labeled ribosome complexes (B) Native gel analysis indicating similar magnesium sensitivities for Dom34:Hbs1-mediated formation of peptidyl-tRNA and separation of subunits. (C) Rates of peptidyl-tRNA formation and subunit separation are similar. k_cat values are reported as mean +/− SD (N=3). (D) Native gel analysis reveals distinct products generated from Dom34:Hbs1 treatment of initiation (MX) and elongation (MFX) complexes. Met-tRNA^iMet partitions with 40S subunits, while Met-Phe-peptidyl-tRNA is fully liberated from the ribosome.

Figure 4

Intrinsic subunit separation activity associated with eRF1:eRF3. (A) Kinetics of peptidyl-tRNA release stimulated by eRF1(AGQ):eRF3. Symbols: ■ Dom34:Hbs1; ▲, eRF1(AGQ)/eRF3, ▼, mock; ◆, eRF1(AGQ)/eRF3 (GDPNP). (B) Stimulation of subunit separation capacity of eRF1(AGQ) by eRF3. Symbols: ■, Dom34/Hbs1; ▲, eRF1(AGQ)/eRF3; ◆, eRF1(AGQ); ▼, eRF3; ●, mock. (C) Native gel analysis of the codon dependence (UAA vs. CAA) of eRF1(AGQ):eRF3 recycling activity.