Evolutionary patterns of the mitochondrial genome in the Moorish gecko, Tarentola mauritanica

A previous study on the evolutionary patterns of Tarentola mauritanica demonstrated that low levels of mitochondrial diversity observed in the European populations relative to nuclear markers were consistent with a selective sweep hypothesis. In order to unravel the mitochondrial evolutionary history in this European population and two other lineages of T. mauritanica (Iberian and North African clades), variation within 22 nearly complete mitogenomes was analyzed. Surprisingly, each clade seems to have a distinct evolutionary history; with both the European and Iberian clades presenting a decrease of polymorphism, which in the former is consistent with departure from neutrality of the mtDNA (positive or background selection), but in the latter seems to be the result of a bottleneck after a population expansion. The pattern exhibited by the North African clade seems to be a consequence of adaptation to certain mtDNA variants by positive selection.     

tRNA-dependent amide bond–forming enzymes in peptide natural product biosynthesis

In the ribosome-independent biosynthesis of peptide natural products, amino acid building blocks are generally activated in the form of phosphoesters, esters, or thioesters prior to amide bond formation. Following the recent discovery of bacterial enzymes that utilize an aminoacyl ester with a transfer ribonucleic acid (tRNA) in primary metabolism, the number of tRNA-dependent enzymes used in biosynthetic studies of peptide natural products has increased steadily. In this review, we summarize the rapidly growing knowledge base regarding two types of tRNA-dependent enzymes, which are structurally and functionally distinct. Initially, we focus on enzymes with the GCN5-related N-acetyltransferase fold and discuss the catalytic function and aminoacyl-tRNA recognition. Next, newly found peptide-amino acyl tRNA ligases and their ATP-dependent reactions are highlighted.     

The structure of the TrmE GTP-binding protein and its implications for tRNA modification

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

Solution NMR structure of the ribosomal protein RP-L35Ae from Pyrococcus furiosus

The ribosome consists of small and large subunits each composed of dozens of proteins and RNA molecules. However, the functions of many of the individual protomers within the ribosome are still unknown. In this article, we describe the solution NMR structure of the ribosomal protein RP-L35Ae from the archaeon Pyrococcus furiosus. RP-L35Ae is buried within the large subunit of the ribosome and belongs to Pfam protein domain family PF01247, which is highly conserved in eukaryotes, present in a few archaeal genomes, but absent in bacteria. The protein adopts a six-stranded anti-parallel β-barrel analogous to the "tRNA binding motif" fold. The structure of the P. furiosus RP-L35Ae presented in this article constitutes the first structural representative from this protein domain family.     

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr(178), generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. K(m) values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μm, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.