Abstract

Regulation of translation directly controls gene expression levels from mRNA to protein in the translation cycle steps. Ribosome arrest peptides (RAPs) are often conditional modulators interacting with the ribosomal tunnel and induce translational stalling to regulate downstream gene expression in cis to fulfill real-time cellular needs. The ribosomal tunnel also provides a protected environment for initial protein folding. This dissertation's first publication presents a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two alpha-helices connected by an alpha-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50–55 Å of the ribosomal tunnel. The structure reveals how alpha-helix formation directly within the peptidyltransferase center (PTC) of the ribosome interferes with aminoacyl-tRNA (A-tRNA) accommodation, suggesting that for canonical translation, a significant role of the ribosomal tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome. On the other hand, secondary structure formation at the PTC could also be used by the ribosome for specific nascent proteins like RAPs to modulate the rate of translation, which could have significant downstream consequences for co-translational protein targeting and folding. Relief of VemP-mediated ribosome stalling is proposed to result from the translocon pulling force exerted on VemP nascent chain's N-terminal signal sequence. For VemP, force application would inevitably prevent the formation of the extensive secondary structure during translation or lead to an unraveling of any secondary structure that does form within the tunnel, thereby eventually allowing the sterical transition into the induced conformation of the PTC. This work obtained novel insights into the underlying mechanisms of RAP-mediated ribosome force sensing. A translating ribosome can also be stalled on the mRNA owing to defective translational components such as non-stop/no-go mRNAs. To maintain ribosome and protein homeostasis, the ribosome-associated quality control (RQC) system has evolved to rescue the stalled ribosome and release the incomplete nascent protein for degradation through disassembling the translation machinery in eukaryotic cells. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by adding C-terminal alanyl and threonyl residues (CAT tails), whereas Vms1 catalyzes cleavage and release of the peptidyl-tRNA before or after addition of CAT tails. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis. This dissertation's second publication presents structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger, and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA base 73 for nucleolytic cleavage. Moreover, in the pre-state, ABCF-type ATPase Arb1 was found in the ribosomal E-site, which stabilizes the delocalized base 73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. These structural analyses provided mechanistic insights into the interplay of the RQC factors Vms1, Rqc2, and Arb1 and their role in protecting mitochondria from the aggregation of toxic proteins.