Jane E. Jackman

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R‐3‐hydroxyacyl‐ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42°C. This was accompanied by a dramatic increase in the amount of UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse‐chase experiments and in vitro assays with purified components showed that FtsH, the AAA‐type membrane‐bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl‐ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R‐3‐hydroxyacyl‐ACP pool, is regulated by FtsH.

Transfer RNA modifications: nature's combinatorial chemistry playground

Following synthesis, tRNAs are peppered by numerous chemical modifications which may differentially affect a tRNAs structure and function. Although modifications affecting the business ends of a tRNA are predictably important for cell viability, a majority of modifications play more subtle structural roles that can affect tRNA stability and folding. The current trend is that modifications act in concert and it is in the context of the specific sequence of a given tRNA that they impart their differing effects. Recent developments in the modification field have highlighted the diversity of modifications in tRNA. From these, the combinatorial nature of modifications in explaining previously described phenotypes derived from their absence has emerged as a growing theme. WIREs RNA 2013, 4:35–48. doi: 10.1002/wrna.1144

Antibacterial and anti-inflammatory agents that target endotoxin

Antibiotic-resistant bacterial infections are a major clinical problem. Lipid A, the active part of lipopolysaccharide endotoxins in Gram-negative bacteria, is an intriguing target for new antibacterial and anti-inflammatory agents. Inhibition of lipid A biosynthesis kills most Gram-negative bacteria, increases bacterial permeability to antibiotics and decreases endotoxin production.

Template-dependent 3′–5′ nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life

The presence of an additional 5′ guanosine residue (G-1) is a unique feature of tRNAHis. G-1 is incorporated posttranscriptionally in eukarya via an unusual 3′–5′ nucleotide addition reaction catalyzed by the tRNAHis guanylyltransferase (Thg1). Yeast Thg1 catalyzes an unexpected second activity: Watson–Crick-dependent 3′–5′ nucleotide addition that occurs in the opposite direction to nucleotide addition by all known DNA and RNA polymerases. This discovery led to the hypothesis that there are alternative roles for Thg1 family members that take advantage of this unusual enzymatic activity. Here we show that archaeal homologs of Thg1 catalyze G-1 addition, in vitro and in vivo in yeast, but only in a templated reaction, i.e. with tRNAHis substrates that contain a C73 discriminator nucleotide. Because tRNAHis from archaea contains C73, these findings are consistent with a physiological function for templated nucleotide addition in archaeal tRNAHis maturation. Moreover, unlike yeast Thg1, archaeal Thg1 enzymes also exhibit a preference for template-dependent U-1 addition to A73-containing tRNAHis. Taken together, these results demonstrate that Watson–Crick template-dependent 3′–5′ nucleotide addition is a shared catalytic activity exhibited by Thg1 family members from multiple domains of life, and therefore, that this unusual reaction may constitute an ancestral activity present in the earliest members of the Thg1 enzyme family.

DISRUPTION OF THE ACTIVE SITE SOLVENT NETWORK IN CARBONIC ANHYDRASE II DECREASES THE EFFICIENCY OF PROTON TRANSFER

The importance of maintaining the active site water network for efficient proton transfer was investigated by substituting amino acids of varying size at position 65 in carbonic anhydrase II (including four amino acids found in other CA isozymes, F, L, S, and T, and two amino acids that do not occur naturally at position 65, G and H) and measuring the rate constants for the proton transfer reactions in the variant carbonic anhydrases. Intramolecular proton transfer between zinc-bound water and H64 is significantly inhibited by the introduction of bulky residues at position 65; kcat for CO2 hydration decreases up to 26-fold, comparable to the observed decrease in intramolecular proton transfer caused by removal of H64 [Tu, C., Silverman, D. N., Forsman, C., Jonsson, B.-H., & Lindskog, S. (1989) Biochemistry 28, 7913-7918]. Intermolecular proton transfer between protonated H64 and external buffer is also inhibited, although to a lesser degree. Furthermore, an alternative proton transfer pathway, consisting of an active site solvent-mediated proton transfer from zinc-water to imidazole buffer, is inhibited in the A65F, A65L, and A65H CAII variants. Therefore, the active solvent bridge between zinc-bound water and H64 is disrupted by substitutions at position 65. The inhibition of proton transfer reactions correlates with the disruption of the crystallographically observed solvent network in the CA active site and rotation of the proton acceptor, H64 [Scolnick, L. R., & Christianson, D. W. (1996) Biochemistry 35, 16429-16434], suggesting that this solvent network, including water molecules 292, 264, and 369, or a structurally related network, forms the proton transfer pathway in CAII for both intramolecular proton transfer and stimulation of proton transfer in imidazole buffers.

Detection and discovery of RNA modifications using microarrays

Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m22G, m1A and m26A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex.

tRNAHis guanylyltransferase (THG1), a unique 3'-5' nucleotidyl transferase, shares unexpected structural homology with canonical 5'-3' DNA polymerases.

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNAHis guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases.

TRMT10A dysfunction is associated with abnormalities in glucose homeostasis, short stature and microcephaly

Background Trm10 is a tRNA m1G9 methyltransferase, which in yeast modifies 12 different tRNA species, yet is considered non-essential for viability under standard growth conditions. In humans, there are three Trm10 orthologs, one mitochondrial and two presumed cytoplasmic. A nonsense mutation in one of the cytoplasmic orthologs (TRMT10A) has recently been associated with microcephaly, intellectual disability, short stature and adolescent onset diabetes. Methods and results The subjects were three patients who suffered from microcephaly, intellectual disability, short stature, delayed puberty, seizures and disturbed glucose metabolism, mainly hyperinsulinaemic hypoglycaemia. A homozygous Gly206Arg (G206R) mutation in the TRMT10A gene was identified using whole exome sequencing. The mutation segregated in the family and was absent from large control cohorts. Determination of the methylation activity of the expressed wild-type (WT) and variant TRMT10A enzymes with transcripts of 32P -tRNAGlyGCC as a substrate revealed a striking defect (<0.1% of WT activity) for the variant enzyme. The binding affinity of the G206R variant enzyme to tRNA, determined by fluorescence anisotropy, was similar to that of the WT enzyme. Conclusions The completely abolished m1G9 methyltransferase activity of the mutant enzyme is likely due to significant defects in its ability to bind the methyl donor S-adenosyl methionine. We propose that TRMT10A deficiency accounts for abnormalities in glucose homeostasis initially manifesting both ketotic and non-ketotic hypoglycaemic events with transition to diabetes in adolescence, perhaps as a consequence of accelerated β cell apoptosis. The seizure disorder and intellectual disability are probably secondary to mutant gene expression in neuronal tissue.

tRNA 5′-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea

The tRNAHis guanylyltransferase (Thg1) family comprises a set of unique 3′–5′ nucleotide addition enzymes found ubiquitously in Eukaryotes, where they function in the critical G−1 addition reaction required for tRNAHis maturation. However, in most Bacteria and Archaea, G−1 is genomically encoded; thus post-transcriptional addition of G−1 to tRNAHis is not necessarily required. The presence of highly conserved Thg1-like proteins (TLPs) in more than 40 bacteria and archaea therefore suggests unappreciated roles for TLP-catalyzed 3′–5′ nucleotide addition. Here, we report that TLPs from Bacillus thuringiensis (BtTLP) and Methanosarcina acetivorans (MaTLP) display biochemical properties consistent with a prominent role in tRNA 5′-end repair. Unlike yeast Thg1, BtTLP strongly prefers addition of missing N+1 nucleotides to 5′-truncated tRNAs over analogous additions to full-length tRNA (kcat/KM enhanced 5–160-fold). Moreover, unlike for −1 addition, BtTLP-catalyzed additions to truncated tRNAs are not biased toward addition of G, and occur with tRNAs other than tRNAHis. Based on these distinct biochemical properties, we propose that rather than functioning solely in tRNAHis maturation, bacterial and archaeal TLPs are well-suited to participate in tRNA quality control pathways. These data support more widespread roles for 3′–5′ nucleotide addition reactions in biology than previously expected.

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC).

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.