Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNAGln is primarily synthesized by first forming Glu-tRNAGln, followed by conversion to Gln-tRNAGln by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNAGln and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNAGln. These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNAGln, consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNAGln synthesis

A genome wide dosage suppressor network reveals genomic robustness

Genomic robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of genomic robustness in budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggests a surprising degree of functional plasticity of macromolecular complexes, and the existence of numerous degenerate pathways for circumventing the effects of potentially lethal mutations. These results imply that organisms and cancer are likely able to exploit the genomic robustness properties, due the persistence of cryptic gene and pathway functions, to generate variation and adapt to selective pressures

Specific Codon Pairs Inhibit Translation in Yeast and Act by Distinct Mechanisms

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2017.​Translation shapes the proteome and is highly regulated to ensure synthesis of functional proteins. The choice of synonymous codons used to encode a polypeptide modulates translation efficiency and co-translational protein folding, but the identities and properties of codons or codon combinations that impair translation were unknown. In a systematic analysis of the effects of 59/61 sense codons, our lab identified the Arg CGA codon as strongly inhibitory. Furthermore, pairs of CGA codons (CGA-CGA) are stronger inhibitors of translation than single CGA codons. Thus, we reasoned that there could be other pairs of codons that inhibit translation. To identify such inhibitory pairs in the yeast Saccharomyces cerevisiae, we employed a high throughput method to screen a library of over 35,000 GFP variants, in which three adjacent codons were randomized, using fluorescence-activated cell sorting (FACS) and deep-sequencing. We identified 17 codon pairs that substantially reduce GFP expression, showing that the pair, rather than the nucleotide sequence, mRNA structure, or individual codons, is responsible for inhibition. These pairs affect translation since their inhibitory effects are suppressed by tRNA. Specific types of wobble decoding are key to inhibition since an exact base pairing tRNA is a more effective suppressor than a wobble base pairing tRNA. Twelve of the most inhibitory codon pairs are slowly translated in native yeast genes, thus linking slow translation to reduced expression. Moreover, since the order of the codons is critical for inhibition, we infer that an interplay between tRNAs at adjacent sites in the ribosome regulates translation. infer that these codon pairs work by different mechanisms. Inhibition by three pairs, including CGA-CGA, depends upon the ribosomal Asc1 protein and their effects are mediated by the ribosome quality control (RQC) complex. Since inhibition by nine other inhibitory pairs is not dependent upon Asc1, I set up a genetic selection to identify genes that modulate their effects

Selection, Identification, and Characterization of Codon Pairs that Inhibit Translation and the Development of the RNA-ID Method to Measure the Effects of Cis-Regulatory Elements

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2013.It has been known for over 30 years that synonymous codon choice regulates translation, but the characteristics of codons that inhibit translation, and the factors that restrict their function, are unknown. In a systematic screen of 59 of 61 sense codons, our lab identified the arginine CGA codon as strongly inhibitory, and then determined that CGA inhibition was primarily due to A·I wobble decoding. Furthermore, I found that adjacent CGA codons are synergistically inhibitory relative to separated CGA codons, which presents the possibility that combinations of non-identical codons may regulate translation in ways that are not predictable from their individual codon behavior. To search for inhibitory codon combinations among the 3,721 codon pairs and 226,981 codon triplets, I developed the RNA-ID method to screen for cis-regulatory elements. RNA-ID utilizes a fluorescence-based, integrated reporter and coupled to flow cytometry to evaluate effects of sequences on expression in Saccharomyces cerevisiae. This method is useful because insertion of test sequences, using ligation independent cloning, is simple, expression is detectable over a 250-fold range, measurements are quantitative and dose-dependent, separation of specific populations is nearly complete, and results from a single sequence are reproducible. To find inhibitory codon combinations, a library of sequences, consisting of random 9 nucleotide inserts, was evaluated using RNA-ID. Only a small fraction of yeast (<0.2%) exhibited GFP/RFP at 6-10% the maximal level, indicating that very few sequences inhibit expression to this degree. Ninety-two strains from this group were studied. Five novel sequences that inhibit translation where identified based on suppression of the expression defect by appropriate tRNAs. The inhibitory sequences all contain at least one codon decoded by wobble, and in each case, the suppression of the inhibitory effect requires expression of a mutated, exact base-pairing tRNA. Thus, I conclude that wobble decoding is likely to be a critical factor in their inhibition of translation. In two cases, inhibition depends upon a pair of adjacent codons and upon the order of these codons, thus indicating that the intact codon pair is the inhibitory unit, consistent with the idea that they act within a single round of translation

Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22, and the 5’-3’ exonucleases Rat1 and Xrn1

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.tRNAs are extremely stable RNA molecules that are exquisitely formed for their specific roles in translation. Formation of fully functional tRNA in all organisms involves multiple processing steps, including the addition of numerous modifications, 25 of which are found in the yeast Saccharomyces cerevisiae. This work describes a novel tRNA degradation pathway that acts on several mature tRNA species lacking combinations of modifications, underscoring the importance of tRNA modifications for tRNA stability and function in vivo. Recent work in this laboratory showed that tRNAVal(AAC) lacking the m7G46 and m5C49 modifications due to mutation of TRM8 and TRM4 is rapidly degraded at 37 °C by a novel mechanism. It is demonstrated here that loss of tRNAVal(AAC) causes the temperature sensitive defect of trm8-Δ trm4-Δ mutants. Furthermore, degradation of hypomodified tRNAVal(AAC) is accompanied by loss of its aminoacylation and occurs at the level of mature tRNA. Analysis of spontaneous suppressors of the temperature sensitive phenotype of trm8-Δ trm4-Δ mutants demonstrates that this mature tRNA degradation pathway is mediated by Met22, and the 5’-3’ exonucleases Rat1 and Xrn1. Mutation or deletion of MET22 completely rescues the temperature sensitive growth defect of the trm8-Δ trm4-Δ strain, and prevents both degradation and loss of aminoacylation of tRNAVal(AAC). Loss of Met22 function has previously been linked to inhibition of Rat1 and Xrn1 due to accumulation of v the Met22 substrate pAp. Consistent with this, mutation of the essential RAT1 gene together with deletion of XRN1 also completely stabilizes both the levels and aminoacylation of hypomodified tRNAVal(AAC) at 37 °C, and rescues growth of trm8-Δ trm4-Δ mutants. Furthermore, this tRNA degradation pathway acts on multiple mature tRNA species, and might play a wider role in turnover of mature tRNA in vivo. It has recently been shown that levels of tRNASer(CGA) and tRNASer(UGA) lacking Um44 and ac4C12 decrease in a temperature dependent fashion, leading to temperature sensitive growth of the trm44-Δ tan1-Δ strain. It is shown here that deletion of MET22 also prevents degradation of mature hypomodified tRNASer(CGA) and tRNASer(UGA), and that mutation of RAT1 in the trm44-Δ tan1-Δ background suppresses the temperature sensitive defect of this strain

Identification of yeast tRNA Um44 2′-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNASer species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um44 is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNASer in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNASer Um44 2′-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-Δ strain lacks 2′-O-methyltransferase activity and has undetectable levels of Um44 in its tRNASer and by showing that Trm44 purified from Escherichia coli 2′-O-methylates U44 of tRNASer in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um44 in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-Δ mutants have no detectable growth defect, TRM44 is required for survival at 33°C in a tan1-Δ mutant strain, which lacks ac4C12 in tRNASer and tRNALeu. At nonpermissive temperature, a trm44-Δ tan1-Δ mutant strain has reduced levels of tRNASer(CGA) and tRNASer(UGA), but not other tRNASer or tRNALeu species. The trm44-Δ tan1-Δ growth defect is suppressed by addition of multiple copies of tRNASer(CGA) and tRNASer(UGA), directly implicating these tRNASer species in this phenotype. The reduction of specific tRNASer species in a trm44-Δ tan1-Δ mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control

A Systematic Analysis of Codon Effects on Translation Efficiency: Identification and Mechanistic Examination of the Inhibitory Effects of the Arginine Codon CGA

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2011.The particular choice of codons used to encode a polypeptide influences translational efficiency, accuracy, reading frame maintenance, and nascent polypeptide folding. Although the codons that are implicated in efficient translation are well established, there has been no comprehensive analysis of individual codon effects on expression in any organism. To systematically study the effects of codons on translation efficiency, 10 repeats of each codon were inserted upstream of the firefly luciferase reporter in two positions, either near the translation start or in the middle of a fusion construct. The arginine codon CGA was identified as a strong inhibitor of translation efficiency. CGA-mediated inhibition of expression occurs with as few as three arginine residues, acts on at least three downstream genes, and is dependent upon CGA codon dosage. Two CGA codons result in a 2-fold reduction in expression and 5 CGA codons result in a 25-fold reduction, which are surprising results in view of the prevailing idea that individual codons effects on expression are relatively small. Moreover, CGA-mediated inhibition results from inefficient translation, since an anticodon-matched isoaccepting tRNA species efficiently rescues expression. This result implies that inefficient I•A wobble decoding, not limiting tRNA amount, is responsible for CGA codon effects. Thus, decoding interactions within the ribosome modulate the efficiency of translation. An examination of the effects of CGA codon repeats on both mRNA and protein species supports a model in which CGA codons elicit ribosome stalling during translation. First, insertion of CGA codons results in a new RNA, whose properties are consistent with cleavage near the CGA codons. Similar RNA cleavage products are detected when ribosomes stall due to strong RNA secondary structures. Second, the presence of CGA codons results in a truncated polypeptide, the amount of which is dramatically increased in the absence of Ltn1p, an E3 ubiquitin ligase that recognizes ribosomes stalled due to the absence of a stop codon. Thus, the presence of the polypeptide and its susceptibility to Ltn1-mediated degradation are expected if ribosomes stall at CGA codons. These results provide the basis for determining the mechanisms by which CGA codons exert their effects on translation