No abstract

To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final noncognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre-and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNA Leu in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pretransfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.

The microbial composition and in vitro fermentation characteristics of human milk oligosaccharides (HMO), lacto-N-neotetraose (LNnT), a 2:1 mixture of polydextrose (PDX) and galactooligosaccharides (GOS), and short-chain fructooligosaccharides (scFOS) by pooled ascending colonic microbiota from 9- and 17-d-old formula-fed (FF) and sow-reared (SR) piglets were assessed. pH change and gas, SCFA, and lactate production were determined after 0, 2, 4, 8, and 12 h of incubation. In most donor groups, the pH change was greater for scFOS fermentation and lower for PDX/GOS than for other substrates. LNnT fermentation produced larger amounts of gas, total SCFA, acetate, and butyrate than did the other substrates, whereas HMO and scFOS produced higher amounts of propionate and lactate, respectively. In general, pH change, total SCFA, acetate, and propionate production were greater in pooled inoculum from FF and 9-d-old piglets, whereas SR-derived inoculum produced higher amounts of butyrate and lactate after 4 h fermentation. Gut microbiota were assessed by 16S ribosomal RNA V3 gene denaturing gradient gel electrophoresis analysis and real-time qPCR. Microbial structures differed among the 4 groups before fermentation, with higher counts of Bifidobacterium in SR piglets and higher counts of Clostridium cluster IV, XIVa, and Bacteroides vulgatus in FF piglets. Lactobacillus counts were higher in 9-d-old piglets than in 17-d-old piglets, regardless of diet. Bifidobacterium, Bacteroides, and clostridial species increased after 8 and 12 h fermentation on most substrates. In summary, piglet diet and age affect gut microbiota, leading to different fermentation patterns. HMO have potential prebiotic effects due to their effects on SCFA production and microbial modulation.