Wyosine (imG) and its derivatives such as wybutosine (yW) are found at position 37 of phenylalanine-specific transfer RNA (tRNA(Phe)), 3' adjacent to the anticodon in Eucarya and Archaea. In Saccharomyces cerevisiae, formation of yW requires five enzymes acting in a strictly sequential order: Trm5, Tyw1, Tyw2, Tyw3, and Tyw4. Archaea contain wyosine derivatives, but their diversity is greater than in eukaryotes and the corresponding biosynthesis pathways still unknown. To identify these pathways, we analyzed the phylogenetic distribution of homologues of the yeast wybutosine biosynthesis proteins in 62 archaeal genomes and proposed a scenario for the origin and evolution of wyosine derivatives biosynthesis in Archaea that was partly experimentally validated. The key observations were 1) that four of the five wybutosine biosynthetic enzymes are ancient and may have been present in the last common ancestor of Archaea and Eucarya, 2) that the variations in the distribution pattern of biosynthesis enzymes reflect the diversity of the wyosine derivatives found in different Archaea. We also identified 7-aminocarboxypropyl-demethylwyosine (yW-86) and its N4-methyl derivative (yW-72) as final products in tRNAs of several Archaea when these were previously thought to be only intermediates of the eukaryotic pathway. We confirmed that isowyosine (imG2) and 7-methylwyosine (mimG) are two archaeal-specific guanosine-37 derivatives found in tRNA of both Euryarchaeota and Crenarchaeota. Finally, we proposed that the duplication of the trm5 gene in some Archaea led to a change in function from N1 methylation of guanosine to C7 methylation of 4-demethylwyosine (imG-14).

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SignificanceProtein and mRNA expression are in most cases poorly correlated, which suggests that the posttranscriptional regulatory program of a cell is an important component of gene expression. This regulatory network is still poorly understood, including how RNA structure quantitatively contributes to translational control. We present here a series of structural and functional experiments that together allow us to derive a quantitative, structure-dependent model of translation that accurately predicts translation efficiency in reporter assays and primary human tissue for a complex and medically important protein, α-1-antitrypsin. Our model demonstrates the importance of accurate, experimentally derived RNA structural models partnered with Kozak sequence information to explain protein expression and suggests a strategy by which α-1-antitrypsin expression may be increased in diseased individuals.

Folding to a well-defined conformation is essential for the function of structured ribonucleic acids (RNAs) like the ribosome and tRNA. Structured elements in the untranslated regions (UTRs) of specific messenger RNAs (mRNAs) are known to control expression. The importance of unstructured regions adopting multiple conformations, however, is still poorly understood. High-resolution SHAPE-directed Boltzmann suboptimal sampling of the Homo sapiens Retinoblastoma 1 (RB1) 5 ′ UTR yields three distinct conformations compatible with the experimental data. Private single nucleotide variants (SNVs) identified in two patients with retinoblastoma each collapse the structural ensemble to a single but distinct well-defined conformation. The RB1 5 ′ UTRs from Bos taurus (cow) and Trichechus manatus latirostris (manatee) are divergent in sequence from H. sapiens (human) yet maintain structural compatibility with high-probability base pairs. SHAPE chemical probing of the cow and manatee RB1 5 ′ UTRs reveals that they also adopt multiple conformations. Luciferase reporter assays reveal that 5 ′ UTR mutations alter RB1 expression. In a traditional model of disease, causative SNVs disrupt a key structural element in the RNA. For the subset of patients with heritable retinoblastoma-associated SNVs in the RB1 5 ′ UTR, the absence of multiple structures is likely causative of the cancer. Our data therefore suggest that selective pressure will favor multiple conformations in eukaryotic UTRs to regulate expression.

With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.

Archaeosine (G+) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G+ precursor, 7-cyano-7-deazaguanine (preQ0), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G+ by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G+ lack ArcS homologs. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: 1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ0; 2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ0 to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G+ in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G+ in Crenarchaeota.

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The presence of the 7-deazaguanosine derivative archaeosine (G ؉ ) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ 0 ), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ 0 -tRNA. The enzymes responsible for the biosynthesis of preQ 0 were recently identified, but the enzyme(s) catalyzing the conversion of preQ 0 -tRNA to G ؉ -tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structurebased alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii ⌬tgtA2 derivative and demonstrated that tRNA from this strain lacks G ؉ and instead accumulates preQ 0 . We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ 0 -tRNA to G ؉ -tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.