Analysis of sRNAs and Their mRNA Targets in Sinorhizobium meliloti: Focus on Half-Life Determination.

Regulation of gene expression at the level of RNA and/or by regulatory RNA is an integral part of the regulatory circuits in all living cells. In bacteria, transcription and translation can be coupled, enabling regulation by transcriptional attenuation, a mechanism based on mutually exclusive structures in nascent mRNA. Transcriptional attenuation gives rise to small RNAs that are well suited to act in trans by either base pairing or ligand binding. Examples of 5'-UTR-derived sRNAs in the alpha-proteobacterium Sinorhizobium meliloti are the sRNA rnTrpL of the tryptophan attenuator and SAM-II riboswitch sRNAs. Analyses addressing RNA-based gene regulation often include measurements of steady-state levels and of half-lives of specific sRNAs and mRNAs. Using such measurements, recently we have shown that the tryptophan attenuator responds to translation inhibition by tetracycline and that SAM-II riboswitches stabilize RNA. Here we discuss our experience in using alternative RNA purification methods for analysis of sRNA and mRNA of S. meliloti. Additionally, we show that other translational inhibitors (besides tetracycline) also cause attenuation giving rise to the rnTrpL sRNA. Furthermore, we discuss the importance of considering RNA stability changes under different conditions and describe in detail a robust and fast method for mRNA half-life determination. The latter includes rifampicin treatment, RNA isolation using commercially available columns, and mRNA analysis by reverse transcription followed by quantitative PCR (RT-qPCR). The latter can be performed as a one-step procedure or in a strand-specific manner using the same commercial kit and a spike-in transcript as a reference.

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase.

The berberine bridge enzyme from the California poppy Eschscholzia californica (EcBBE) catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE-like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of EcBBE in Physcomitrella patens (PpBBE1), the most basal plant harboring a BBE-like gene. Here, we report the biochemical, structural, and in vivo characterization of PpBBE1. Our studies revealed that PpBBE1 is structurally and biochemically very similar to EcBBE. In contrast to EcBBE, we found that PpBBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, PpBBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure-guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of PpBBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE-like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization. DATABASE: Structural data are available in the PDB under the accession numbers 6EO4 and 6EO5.