Structural and Functional Insights into Saccharomyces cerevisiae Tpa1, a Putative Prolylhydroxylase Influencing Translation Termination and Transcription

Abstract

Efficiency of translation termination relies on the specific recognition of the three stop codons by the eukaryotic translation termination factor eRF1. To date only a few proteins are known to be involved in translation termination in eukaryotes. Saccharomyces cerevisiae Tpa1, a largely conserved but uncharacterized protein, has been described to associate with a messenger ribonucleoprotein complex located at the 3 end of mRNAs that contains at least eRF1, eRF3, and Pab1. Deletion of the TPA1 gene results in a decrease of translation termination efficacy and an increase in mRNAs half-lives and longer mRNA poly(A) tails. In parallel, Schizosaccharomyces pombe Ofd1, a Tpa1 ortholog, and its partner Nro1 have been implicated in the regulation of the stability of a transcription factor that regulates genes essential for the cell response to hypoxia. To gain insight into Tpa1/ Ofd1 function, we have solved the crystal structure of S. cerevisiae Tpa1 protein. This protein is composed of two equivalent domains with the double-stranded ␤-helix fold. The N-terminal domain displays a highly conserved active site with strong similarities with prolyl-4-hydroxylases. Further functional studies show that the integrity of Tpa1 active site as well as the presence of Yor051c/Ett1 (the S. cerevisiae Nro1 ortholog) are essential for correct translation termination. In parallel, we show that Tpa1 represses the expression of genes regulated by Hap1, a transcription factor involved in the response to levels of heme and oxygen. Altogether, our results support that Tpa1 is a putative enzyme acting as an oxygen sensor and influencing several distinct regulatory pathways.Protein synthesis is a complex process performed by ribosomes, translation factors, and amino-acyl tRNAs that act synergistically to translate mRNAs into corresponding proteins. The mechanism of translation of mRNAs into protein can be divided into three major steps; they are initiation, elongation, and termination. In eukaryotes, translation initiation relies on the formation of a stable closed-loop structure bringing the 5Ј m 7 G cap and the 3Ј poly(A) tail of a single mRNA in proximity and requires at least 11 initiation factors (1, 2). During elongation, the mRNA codon present in the ribosomal A site is recognized by a cognate amino-acyl tRNA associated with elongation factor 1A. The nascent polypeptide chain is then transferred from the tRNA present in the P-site to the amino acid of the A-site tRNA. Subsequently, elongation factor 2 induces translocation of peptidyl-tRNA from the A-to the P-site allowing the next elongation step to proceed. The entrance of one of the three stop codons (UAA, UAG, or UGA) in the A-site triggers translation termination. In contrast with other codons, stop codons are not recognized by cognate tRNAs but by proteins known as class I release factors (RF1 6 and RF2 in bacteria and eRF1 in eukaryotes). These are also directly involved in the hydrolysis of the ester bond connecting the newly synthesized polypeptide chain to the tRNA located...

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

“…S1 B and C). Given that S. cerevisiae Tpa1p and S. pombe Ofd1 have putative active sites in the N-terminal of their two DSBH domains that possess similarity to PHD2 (15,23), we tested whether Sud1 plays a role in the HIF-dependent transcriptional response to hypoxia by observing the effects of Sud1 knockdown on HIF/Sima-dependent transcription in the embryonic tracheal system. Embryos expressing sud1 RNAi failed to modulate a HIF-dependent transcriptional reporter, whereas, as expected, embryos that express an RNAi targeting the prolyl-4-hydroxylase gene fatiga displayed strong upregulation of the same reporter under mild hypoxic conditions, providing a positive control for the assay (Fig.…”

“…We identified CG44254, a highly conserved gene that is distantly related to oxygen sensing PHDs (14), as necessary for normal growth and mRNA translation in the fly. This gene, which we have termed sudestada1 (sud1) after a wind that blows across the southeastern coast of South America, is highly conserved from yeast to humans; homologous genes in Saccharomyces cerevisiae (TPA1) (15,16), Schizosaccharomyces pombe (Ofd1) (17,18), and Homo sapiens (OGFOD1) (19,20) have been implicated in translation termination, oxygen-dependent regulation of the transcription factor Sre1N, and translational stresses responses, respectively.…”

Fungal Morphogenesis

“…Both Ofd1 proteins have two functional domains: an amino-termi-nal dioxygenase domain with a conserved Fe 2þ binding motif essential for O 2 sensing and a carboxy-terminal domain that promotes protein degradation. The amino-terminal domain inhibits the degradative activity of the carboxyterminal domain in hypoxia (Hughes and Espenshade 2008;Henri et al 2010). The closest structural homolog to the amino-terminal dioxygenase domain is human PHD2 cat , a prolyl 4-hydroxylase that acts as an oxygen-sensing component and hydroxylates the hypoxia-inducible transcription factor HIF1a in the presence of oxygen, thus leading to its degradation by the proteasome (Schofield and Ratcliffe 2005;Ozer and Bruick 2007;Henri et al 2010).…”

“…The amino-terminal domain inhibits the degradative activity of the carboxyterminal domain in hypoxia (Hughes and Espenshade 2008;Henri et al 2010). The closest structural homolog to the amino-terminal dioxygenase domain is human PHD2 cat , a prolyl 4-hydroxylase that acts as an oxygen-sensing component and hydroxylates the hypoxia-inducible transcription factor HIF1a in the presence of oxygen, thus leading to its degradation by the proteasome (Schofield and Ratcliffe 2005;Ozer and Bruick 2007;Henri et al 2010). Ofd1 orthologs with sequence conservation in both amino-and carboxy-terminal domains are found only in fungi, including C. neoformans and A. fumigatus.…”

Structural and functional analysis of Nro1/Ett1: a protein involved in translation termination in S. cerevisiae and in O2-mediated gene control in S. pombe

“…Tpa1 is another protein that was implicated in modulating translation termination efficiency in S. cerevisiae by reducing stop-codon read-through (Keeling et al 2006;Henri et al 2010). In addition, this protein was shown to interact with Pab1, eRF1, and eRF3 and hence proposed to be part of an mRNP complex associated at the mRNA 39 end (Keeling et al 2006).…”